Novel glucose derivative, and cell imaging method and imaging agent using said derivative

ABSTRACT

Provided is a glucose derivative, which is taken into cells via a membrane sugar transport system and is represented by formula (1). Also provided are an imaging agent and an imaging method for a cell or intracellular molecule using said glucose derivative. Further provided are a method for detecting cancer cells with good accuracy using said glucose derivative and an imaging agent to be used in said method. More specifically provided are D-glucose derivatives and L-glucose derivatives in which glucose is bound to the 7-position of a fluorescent molecular group with a coumarin backbone or a quinolone backbone. Also provided are a cell imaging agent and imaging method using the derivative. A cancer cell imaging agent and imaging method using the L-glucose derivative is also provided. G is a group selected from formulas (G1)-(G4) below.

TECHNICAL FIELD

The present invention relates to a novel glucose derivative. The presentinvention also relates to a cell imaging method and imaging agent usingthe novel glucose derivative. The present invention further relates to amethod for detecting and/or imaging cancer cells using the novel glucosederivative, and an imaging agent therefor.

BACKGROUND ART

Molecular imaging has been actively conducted which performs imaging forvisualization of cells, particularly, living cells, or imaging forvisualization targeting molecules in living bodies to reveal molecularkinetics, intermolecular interaction, and molecular positionalinformation, leading to the elucidation of the mechanisms of lifescience or drug discovery screening. Particularly, researches have alsobeen actively made to detect cancer cells or cancer sites by visualizingabnormal cells, for example, cancer cells.

The group of the present inventors have proposed a method using greenfluorescence-emitting2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-D-glucose(2-NBDG) in which a N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino group isbound as a fluorophore to the 2-position of D-deoxyglucose, as a methodthat can be used in research on the dynamic uptake process of D-glucoseby live cells, and have demonstrated the usefulness of the method usingvarious mammalian cells (Non Patent Literature 1).

This method utilizes 2-NBDG's property of being selectively taken intolive cells and can quantitatively determine dynamic activity as to thecellular uptake of D-glucose by monitoring change in fluorescenceintensity caused by the uptake. Therefore, this method is highlyregarded by researchers around the world as a breakthrough for studyinghow organisms take D-glucose into cells and utilize the D-glucose, andis now accepted as a standard protocol indispensable in this researchfield (Non Patent Literature 2).

In the history of the previous development of fluorescent D-glucosederivatives, 2-NBDG and 6-NBDG which are D-glucose derivatives bound toa green fluorescent group NBD are the only substances that have beeninternationally accepted as being transported into cells via a glucosetransporter GLUT and subjected to various replication studies.Furthermore, 2-NBDG is the only molecule that has been found to be takeninto cells and then phosphorylated like FDG that has been used in thePET examination of cancers. Therefore, since long ago, it has beensuggested that 2-NBDG can be utilized not only for the purposes of basicscience fields but also for application to tumor cell imaging inclinical medicine (Non Patent Literature 3, Patent Literature 1, etc.).Also, many attempts have been made to apply 2-NBDG to the diagnosticimaging of cancers (Non Patent Literatures 4 and 5).

As another compound than 2-NBDG expected to pass through GLUT, theinventors have developed 2-DBDG(2-[N-7-(N′,N′-dimethylaminosulfonyl)benz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-D-glucose)which is a molecule in which DBD that is an analog of NBD is bound toD-glucose, and have reported the properties of this compound (PatentLiterature 2). Even though 2-DBDG has a fluorescence wavelength slightlyshifted to the longer wavelength side as compared with 2-NBDG, itlargely overlaps with 2-NBDG in terms of spectra. Thus, when 2-DBDG isused with 2-NBDG at the same time, a problem arises for discriminatingthem from each other.

In fact, D-glucose derivatives bound to various other fluorescentmolecules than NBD have also been proposed so far. Particularly, therehave been developed a molecule bound to a more highly tissue-permeableprobe having the maximum fluorescence in the near-infrared region, amolecule suitable for excitation using a two-photon microscope, amolecule that emits stronger fluorescence than 2-NBDG, and the like.However, because these D-glucose derivative molecules have a much largermolecular size than 2-NBDG, mechanisms underlying their uptake intocells are presumed to be cellular uptake via pathways other than GLUT,for example, uptake by phagocytosis or by internalization in aprotein-bound state (Non Patent Literature 6).

In addition to the molecules having a green, red, or near-infraredfluorescence spectrum, fluorescent glucose derivatives have also beenreported which have a D-glucose bound to a coumarin derivative moleculehaving a blue fluorescence spectrum (Esculin, Fraxin, and compoundsdescribed in WO2012/70024 (Patent Literature 3)). However, thesederivatives are not taken into cells via GLUT. There has been no reporton a molecule that is a sugar derivative having, in its molecule, a bluefluorescent molecular group and passes through GLUT.

As for substances in which a quinoline derivative molecule is bound toglucose, G. Wagner et al. (Non Patent Literature 7) have reported thefollowing substances:

and Roman Kimmel et al. (Non Patent Literature 8) have reported thefollowing substance:

However, all of these substances have a structure where a quinolinederivative is bound to the 1-position of glucose either directly or viaan oxygen atom. It has not been reported that any of them are taken intocells via GLUT.

Meanwhile, the present inventors have proposed compounds in which3-carboxy-6,8-difluoro-7-hydroxycoumarin (Pacific Blue) or3-carboxymethyl-6,8-difluoro-7-hydroxy-4-methylcoumarin (Marina Blue) isbound to glucose via an amide bond, and have filed a patent applicationof PCT/JP2013/076629.

-   Patent Literature 1: U.S. Pat. No. 6,989,140 specification-   Patent Literature 2: WO2010/16587-   Patent Literature 3: WO2012/70024-   Patent Literature 4: WO2012/133688-   Non Patent Literature 1: Yamada K. et al., J. Biol. Chem. 275:    22278-22283, 2000-   Non Patent Literature 2: Yamada K. et al., Nat. Protoc. 2: 753-762,    2007-   Non Patent Literature 3: O'Neil et al., Mol. Imaging Biol. 7:    388-392-   Non Patent Literature 4: Sheth et al., J. Biomed. Opt. 14:    064014-1-8, 2009-   Non Patent Literature 5: Nitin et al., Int. J. Cancer 2009-   Non Patent Literature 6: Cheng Z. et al., Bioconjugate Chem. 17:    662-669, 2006-   Non Patent Literature 7: G. Wagner, et al., Archiv der Pharmazie,    298 (8), 481-491 (1965)-   Non Patent Literature 8: Roman Kimmel, et al., Carbohydr. Res., 345,    768-779 (2010)

SUMMARY OF INVENTION

A purpose of the present invention is to provide a novel glucosederivative, which is taken into cells via a membrane sugar transportsystem. Another purpose of the present invention is to provide animaging method and imaging agent for a cell using such a glucosederivative. Yet another purpose of the present invention is to provide amethod for detecting cancer cells with good accuracy by imaging, and animaging agent for use in the method.

As a result of conducting diligent studies in light of the pointsdescribed above, the present inventors have completed the presentinvention by finding that a novel glucose derivative having a particularstructure is taken into cells via a membrane sugar transport system. Thepresent inventors have completed the present invention by also findingthat cells can be imaged with good accuracy by use of the novel glucosederivative.

The present invention is as follows:

1. A glucose derivative which is selected from a compound represented bythe following formula (1):

wherein X—Y—Z represents O—C═O, NH—C═O, NR₃—C═O, or N═C—OR₄, wherein R₃represents C₁-C₅ alkyl, and R₄ represents C₁-C₅ alkyl; R₁ and R₂ eachindependently represent a group selected from the group consisting ofhydrogen, halogen, C₁-C₅ alkyl, C₂-C₅ alkenyl, C₂-C₅ alkynyl, C₁-C₅haloalkyl, C₁-C₅ alkylamino, cycloalkyl, phenyl, pyridyl, thiophenyl,pyrrolyl, and furanyl; andG represents a group selected from the following formulas (G1) to (G4):

and a salt thereof.2. The glucose derivative according to the above item 1, which is acompound represented by the following formula (2):

wherein X represents O, NH, or NR₃, wherein R₃ represents C₁-C₅ alkyl;andR₁ and R₂ each independently represent a group selected from the groupconsisting of hydrogen, halogen, C₁-C₅ alkyl, C₂-C₅ alkenyl, C₂-C₅alkynyl, C₁-C₅ haloalkyl, C₁-C₅ alkylamino, cycloalkyl, phenyl, pyridyl,thiophenyl, pyrrolyl, and furanyl, or a salt thereof.3. The glucose derivative according to the above item 2, wherein in theformula (2), X is O.4. The glucose derivative according to the above item 1, which is acompound represented by the following formula (3):

wherein R₁ and R₂ each independently represent a group selected from thegroup consisting of hydrogen, halogen, C₁-C₅ alkyl, C₂-C₅ alkenyl, C₂-C₅alkynyl, C₁-C₅ haloalkyl, C₁-C₅ alkylamino, cycloalkyl, phenyl, pyridyl,thiophenyl, pyrrolyl, and furanyl; and R₄ represents C₁-C₅ alkyl,or a salt thereof.5. The glucose derivative according to the above item 1, which is acompound represented by the following formula (4):

wherein X represents O, NH, or NR₃, wherein R₃ represents C₁-C₅ alkyl;andR₁ and R₂ each independently represent a group selected from the groupconsisting of hydrogen, halogen, C₁-C₅ alkyl, C₂-C₅ alkenyl, C₂-C₅alkynyl, C₁-C₅ haloalkyl, C₁-C₅ alkylamino, cycloalkyl, phenyl, pyridyl,thiophenyl, pyrrolyl, and furanyl, or a salt thereof.6. The glucose derivative according to the above item 5, wherein in theformula (4), X is O.7. The glucose derivative according to the above item 1, which is acompound represented by the following formula (5):

wherein R₁ and R₂ each independently represent a group selected from thegroup consisting of hydrogen, halogen, C₁-C₅ alkyl, C₂-C₅ alkenyl, C₂-C₅alkynyl, C₁-C₅ haloalkyl, C₁-C₅ alkylamino, cycloalkyl, phenyl, pyridyl,thiophenyl, pyrrolyl, and furanyl; and R₄ represents C₁-C₅ alkyl,or a salt thereof.8. The glucose derivative according to the above item 1, which isselected from the group consisting of the following compounds:

-   2-deoxy-2-(2-oxo-2H-chromen-7-yl)amino-D-glucose,    2-deoxy-2-(2-oxo-2H-3-methyl-chromen-7-yl)amino-D-glucose,    2-deoxy-2-(2-oxo-2H-3-trifluoromethyl-chromen-7-yl)amino-D-glucose,    2-deoxy-2-(2-oxo-2H-4-methyl-chromen-7-yl)amino-D-glucose,    2-deoxy-2-(2-oxo-2H-4-trifluoromethyl-chromen-7-yl)amino-D-glucose,    2-deoxy-2-(2-oxo-2H-3-phenyl-chromen-7-yl)amino-D-glucose,    2-deoxy-2-(2-oxo-2H-3-(pyridin-2-yl)-chromen-7-yl)amino-D-glucose,    2-deoxy-2-(2-oxo-2H-3-(pyridin-4-yl)-chromen-7-yl)amino-D-glucose,    2-deoxy-2-(2-oxo-2H-3-(thiophen-2-yl)-chromen-7-yl)amino-D-glucose,    2-deoxy-2-(2-oxo-2H-3-(furan-2-yl)-chromen-7-yl)amino-D-glucose,    2-deoxy-2-(2-oxo-2H-3-(pyrrol-2-yl)-chromen-7-yl)amino-D-glucose,    2-deoxy-2-(2-oxo-2H-3-phenyl-4-methyl-chromen-7-yl)amino-D-glucose,    2-deoxy-2-(2-oxo-2H-3-(pyridin-2-yl)-4-methyl-chromen-7-yl)amino-D-glucose,    2-deoxy-2-(2-oxo-2H-3-(pyridin-4-yl)-4-methyl-chromen-7-yl)amino-D-glucose,    2-deoxy-2-(2-oxo-2H-3-(thiophen-2-yl)-4-methyl-chromen-7-yl)amino-D-glucose,    2-deoxy-2-(2-oxo-2H-3-(furan-2-yl)-4-methyl-chromen-7-yl)amino-D-glucose,    2-deoxy-2-(2-oxo-2H-3-(pyrrol-2-yl)-4-methyl-chromen-7-yl)amino-D-glucose,    2-deoxy-2-(2-oxo-2H-3-phenyl-4-trifluoromethyl-chromen-7-yl)amino-D-glucose,    2-deoxy-2-(2-oxo-2H-3-(pyridin-2-yl)-4-trifluoromethyl-chromen-7-yl)amino-D-glucose,    2-deoxy-2-(2-oxo-2H-3-(pyridin-4-yl)-4-trifluoromethyl-chromen-7-yl)amino-D-glucose,    2-deoxy-2-(2-oxo-2H-3-(thiophen-2-yl)-4-trifluoromethyl-chromen-7-yl)amino-D-glucose,    2-deoxy-2-(2-oxo-2H-3-(furan-2-yl)-4-trifluoromethyl-chromen-7-yl)amino-D-glucose,    2-deoxy-2-(2-oxo-2H-3-(pyrrol-2-yl)-4-trifluoromethyl-chromen-7-yl)amino-D-glucose,    2-deoxy-2-(2-oxo-2H-3-iodo-chromen-7-yl)amino-D-glucose,    2-deoxy-2-(2-oxo-2H-4-iodo-chromen-7-yl)amino-D-glucose,    2-deoxy-2-(2-oxo-2H-3-formyl-chromen-7-yl)amino-D-glucose,    2-deoxy-2-(2-oxo-2H-4-formyl-chromen-7-yl)amino-D-glucose,    2-deoxy-2-(2-oxo-2H-3-ethenyl-chromen-7-yl)amino-D-glucose,    2-deoxy-2-(2-oxo-2H-4-ethenyl-chromen-7-yl)amino-D-glucose,    2-deoxy-2-(2-oxo-2H-3-ethynyl-chromen-7-yl)amino-D-glucose,    2-deoxy-2-(2-oxo-2H-4-ethynyl-chromen-7-yl)amino-D-glucose,    2-deoxy-2-(2-oxo-2H-3,4-dimethyl-chromen-7-yl)amino-D-glucose,    2-deoxy-2-(2-oxo-2H-3-acetyl-chromen-7-yl)amino-D-glucose,    2-deoxy-2-(2-oxo-2H-4-acetyl-chromen-7-yl)amino-D-glucose,    2-deoxy-2-(2-oxo-2H-3-cyclopropanyl-chromen-7-yl)amino-D-glucose,    2-deoxy-2-(2-oxo-2H-4-cyclopropanyl-chromen-7-yl)amino-D-glucose,    2-deoxy-2-(2-oxo-2H-3-methyl-4-acetyl-chromen-7-yl)amino-D-glucose,    2-deoxy-2-(2-oxo-2H-3-acetyl-4-methyl-chromen-7-yl)amino-D-glucose,    2-deoxy-2-(2-oxo-2H-3-propenyl-chromen-7-yl)amino-D-glucose,    2-deoxy-2-(2-oxo-2H-4-propenyl-chromen-7-yl)amino-D-glucose,    2-deoxy-2-(2-oxo-1,2-dihydro-quinolin-7-yl)amino-D-glucose,    2-deoxy-2-(2-oxo-1,2-dihydro-3-methyl-quinolin-7-yl)amino-D-glucose,    2-deoxy-2-(2-oxo-1,2-dihydro-3-trifluoromethyl-quinolin-7-yl)amino-D-glucose,    2-deoxy-2-(2-oxo-1,2-dihydro-4-methyl-quinolin-7-yl)amino-D-glucose,    2-deoxy-2-(2-oxo-1,2-dihydro-4-trifluoromethyl-quinolin-7-yl)amino-D-glucose,    2-deoxy-2-(2-oxo-1,2-dihydro-3-phenyl-quinolin-7-yl)amino-D-glucose,    2-deoxy-2-(2-oxo-1,2-dihydro-3-(pyridin-2-yl)-quinolin-7-yl)amino-D-glucose,    2-deoxy-2-(2-oxo-1,2-dihydro-3-(pyridin-4-yl)-quinolin-7-yl)amino-D-glucose,    2-deoxy-2-(2-oxo-1,2-dihydro-3-(thiophen-2-yl)-quinolin-7-yl)amino-D-glucose,    2-deoxy-2-(2-oxo-1,2-dihydro-3-(furan-2-yl)-quinolin-7-yl)amino-D-glucose,    2-deoxy-2-(2-oxo-1,2-dihydro-3-(pyrrol-2-yl)-quinolin-7-yl)amino-D-glucose,    2-deoxy-2-(2-oxo-1,2-dihydro-3-phenyl-4-methyl-quinolin-7-yl)amino-D-glucose,    2-deoxy-2-(2-oxo-1,2-dihydro-3-(pyridin-2-yl)-4-methyl-quinolin-7-yl)amino-D-glucose,    2-deoxy-2-(2-oxo-1,2-dihydro-3-(pyridin-4-yl)-4-methyl-quinolin-7-yl)amino-D-glucose,    2-deoxy-2-(2-oxo-1,2-dihydro-3-(thiophen-2-yl)-4-methyl-quinolin-7-yl)amino-D-glucose,    2-deoxy-2-(2-oxo-1,2-dihydro-3-(furan-2-yl)-4-methyl-quinolin-7-yl)amino-D-glucose,    2-deoxy-2-(2-oxo-1,2-dihydro-3-(pyrrol-2-yl)-4-methyl-quinolin-7-yl)amino-D-glucose,    2-deoxy-2-(2-oxo-1,2-dihydro-3-phenyl-4-trifluoromethyl-quinolin-7-yl)amino-D-glucose,    2-deoxy-2-(2-oxo-1,2-dihydro-3-(pyridin-2-yl)-4-trifluoromethyl-quinolin-7-yl)amino-D-glucose,    2-deoxy-2-(2-oxo-1,2-dihydro-3-(pyridin-4-yl)-4-trifluoromethyl-quinolin-7-yl)amino-D-glucose,    2-deoxy-2-(2-oxo-1,2-dihydro-3-(thiophen-2-yl)-4-trifluoromethyl-quinolin-7-yl)amino-D-glucose,    2-deoxy-2-(2-oxo-1,2-dihydro-3-(furan-2-yl)-4-trifluoromethyl-quinolin-7-yl)amino-D-glucose,    2-deoxy-2-(2-oxo-1,2-dihydro-3-(pyrrol-2-yl)-4-trifluoromethyl-quinolin-7-yl)amino-D-glucose,    2-deoxy-2-(2-oxo-1,2-dihydro-3-iodo-quinolin-7-yl)amino-D-glucose,    2-deoxy-2-(2-oxo-1,2-dihydro-4-iodo-quinolin-7-yl)amino-D-glucose,    2-deoxy-2-(2-oxo-1,2-dihydro-3-formyl-quinolin-7-yl)amino-D-glucose,    2-deoxy-2-(2-oxo-1,2-dihydro-4-formyl-quinolin-7-yl)amino-D-glucose,    2-deoxy-2-(2-oxo-1,2-dihydro-3-ethenyl-quinolin-7-yl)amino-D-glucose,    2-deoxy-2-(2-oxo-1,2-dihydro-4-ethenyl-quinolin-7-yl)amino-D-glucose,    2-deoxy-2-(2-oxo-1,2-dihydro-3-ethynyl-quinolin-7-yl)amino-D-glucose,    2-deoxy-2-(2-oxo-1,2-dihydro-4-ethynyl-quinolin-7-yl)amino-D-glucose,    2-deoxy-2-(2-oxo-1,2-dihydro-3,4-dimethyl-quinolin-7-yl)amino-D-glucose,    2-deoxy-2-(2-oxo-1,2-dihydro-3-acetyl-quinolin-7-yl)amino-D-glucose,    2-deoxy-2-(2-oxo-1,2-dihydro-4-acetyl-quinolin-7-yl)amino-D-glucose,    2-deoxy-2-(2-oxo-1,2-dihydro-3-cyclopropanyl-quinolin-7-yl)amino-D-glucose,    2-deoxy-2-(2-oxo-1,2-dihydro-4-cyclopropanyl-quinolin-7-yl)amino-D-glucose,    2-deoxy-2-(2-oxo-1,2-dihydro-3-acetyl-4-methyl-quinolin-7-yl)amino-D-glucose,    2-deoxy-2-(2-oxo-1,2-dihydro-3-methyl-4-acetyl-quinolin-7-yl)amino-D-glucose,    2-deoxy-2-(2-oxo-1,2-dihydro-3-propenyl-quinolin-7-yl)amino-D-glucose,    2-deoxy-2-(2-oxo-1,2-dihydro-4-propenyl-quinolin-7-yl)amino-D-glucose,    2-deoxy-2-(1-methyl-2-oxo-1,2-dihydro-quinolin-7-yl)amino-D-glucose,    2-deoxy-2-(2-methoxyquinolin-7-yl)amino-D-glucose,    2-deoxy-2-(2-oxo-2H-chromen-7-yl)amino-L-glucose,    2-deoxy-2-(2-oxo-2H-3-methyl-chromen-7-yl)amino-L-glucose,    2-deoxy-2-(2-oxo-2H-3-trifluoromethyl-chromen-7-yl)amino-L-glucose,    2-deoxy-2-(2-oxo-2H-4-methyl-chromen-7-yl)amino-L-glucose,    2-deoxy-2-(2-oxo-2H-4-trifluoromethyl-chromen-7-yl)amino-L-glucose,    2-deoxy-2-(2-oxo-2H-3-phenyl-chromen-7-yl)amino-L-glucose,    2-deoxy-2-(2-oxo-2H-3-(pyridin-2-yl)-chromen-7-yl)amino-L-glucose,    2-deoxy-2-(2-oxo-2H-3-(pyridin-4-yl)-chromen-7-yl)amino-L-glucose,    2-deoxy-2-(2-oxo-2H-3-(thiophen-2-yl)-chromen-7-yl)amino-L-glucose,    2-deoxy-2-(2-oxo-2H-3-(furan-2-yl)-chromen-7-yl)amino-L-glucose,    2-deoxy-2-(2-oxo-2H-3-(pyrrol-2-yl)-chromen-7-yl)amino-L-glucose,    2-deoxy-2-(2-oxo-2H-3-phenyl-4-methyl-chromen-7-yl)amino-L-glucose,    2-deoxy-2-(2-oxo-2H-3-(pyridin-2-yl)-4-methyl-chromen-7-yl)amino-L-glucose,    2-deoxy-2-(2-oxo-2H-3-(pyridin-4-yl)-4-methyl-chromen-7-yl)amino-L-glucose,    2-deoxy-2-(2-oxo-2H-3-(thiophen-2-yl)-4-methyl-chromen-7-yl)amino-L-glucose,    2-deoxy-2-(2-oxo-2H-3-(furan-2-yl)-4-methyl-chromen-7-yl)amino-L-glucose,    2-deoxy-2-(2-oxo-2H-3-(pyrrol-2-yl)-4-methyl-chromen-7-yl)amino-L-glucose,    2-deoxy-2-(2-oxo-2H-3-phenyl-4-trifluoromethyl-chromen-7-yl)amino-L-glucose,    2-deoxy-2-(2-oxo-2H-3-(pyridin-2-yl)-4-trifluoromethyl-chromen-7-yl)amino-L-glucose,    2-deoxy-2-(2-oxo-2H-3-(pyridin-4-yl)-4-trifluoromethyl-chromen-7-yl)amino-L-glucose,    2-deoxy-2-(2-oxo-2H-3-(thiophen-2-yl)-4-trifluoromethyl-chromen-7-yl)amino-L-glucose,    2-deoxy-2-(2-oxo-2H-3-(furan-2-yl)-4-trifluoromethyl-chromen-7-yl)amino-L-glucose,    2-deoxy-2-(2-oxo-2H-3-(pyrrol-2-yl)-4-trifluoromethyl-chromen-7-yl)amino-L-glucose,    2-deoxy-2-(2-oxo-2H-3-iodo-chromen-7-yl)amino-L-glucose,    2-deoxy-2-(2-oxo-2H-4-iodo-chromen-7-yl)amino-L-glucose,    2-deoxy-2-(2-oxo-2H-3-formyl-chromen-7-yl)amino-L-glucose,    2-deoxy-2-(2-oxo-2H-4-formyl-chromen-7-yl)amino-L-glucose,    2-deoxy-2-(2-oxo-2H-3-ethenyl-chromen-7-yl)amino-L-glucose,    2-deoxy-2-(2-oxo-2H-4-ethenyl-chromen-7-yl)amino-L-glucose,    2-deoxy-2-(2-oxo-2H-3-ethynyl-chromen-7-yl)amino-L-glucose,    2-deoxy-2-(2-oxo-2H-4-ethynyl-chromen-7-yl)amino-L-glucose,    2-deoxy-2-(2-oxo-2H-3,4-dimethyl-chromen-7-yl)amino-L-glucose,    2-deoxy-2-(2-oxo-2H-3-acetyl-chromen-7-yl)amino-L-glucose,    2-deoxy-2-(2-oxo-2H-4-acetyl-chromen-7-yl)amino-L-glucose,    2-deoxy-2-(2-oxo-2H-3-cyclopropanyl-chromen-7-yl)amino-L-glucose,    2-deoxy-2-(2-oxo-2H-4-cyclopropanyl-chromen-7-yl)amino-L-glucose,    2-deoxy-2-(2-oxo-2H-3-methyl-4-acetyl-chromen-7-yl)amino-L-glucose,    2-deoxy-2-(2-oxo-2H-3-acetyl-4-methyl-chromen-7-yl)amino-L-glucose,    2-deoxy-2-(2-oxo-2H-3-propenyl-chromen-7-yl)amino-L-glucose,    2-deoxy-2-(2-oxo-2H-4-propenyl-chromen-7-yl)amino-L-glucose,    2-deoxy-2-(2-oxo-1,2-dihydro-quinolin-7-yl)amino-L-glucose,    2-deoxy-2-(2-oxo-1,2-dihydro-3-methyl-quinolin-7-yl)amino-L-glucose,    2-deoxy-2-(2-oxo-1,2-dihydro-3-trifluoromethyl-quinolin-7-yl)amino-L-glucose,    2-deoxy-2-(2-oxo-1,2-dihydro-4-methyl-quinolin-7-yl)amino-L-glucose,    2-deoxy-2-(2-oxo-1,2-dihydro-4-trifluoromethyl-quinolin-7-yl)amino-L-glucose,    2-deoxy-2-(2-oxo-1,2-dihydro-3-phenyl-quinolin-7-yl)amino-L-glucose,    2-deoxy-2-(2-oxo-1,2-dihydro-3-(pyridin-2-yl)-quinolin-7-yl)amino-L-glucose,    2-deoxy-2-(2-oxo-1,2-dihydro-3-(pyridin-4-yl)-quinolin-7-yl)amino-L-glucose,    2-deoxy-2-(2-oxo-1,2-dihydro-3-(thiophen-2-yl)-quinolin-7-yl)amino-L-glucose,    2-deoxy-2-(2-oxo-1,2-dihydro-3-(furan-2-yl)-quinolin-7-yl)amino-L-glucose,    2-deoxy-2-(2-oxo-1,2-dihydro-3-(pyrrol-2-yl)-quinolin-7-yl)amino-L-glucose,    2-deoxy-2-(2-oxo-1,2-dihydro-3-phenyl-4-methyl-quinolin-7-yl)amino-L-glucose,    2-deoxy-2-(2-oxo-1,2-dihydro-3-(pyridin-2-yl)-4-methyl-quinolin-7-yl)amino-L-glucose,    2-deoxy-2-(2-oxo-1,2-dihydro-3-(pyridin-4-yl)-4-methyl-quinolin-7-yl)amino-L-glucose,    2-deoxy-2-(2-oxo-1,2-dihydro-3-(thiophen-2-yl)-4-methyl-quinolin-7-yl)amino-L-glucose,    2-deoxy-2-(2-oxo-1,2-dihydro-3-(furan-2-yl)-4-methyl-quinolin-7-yl)amino-L-glucose,    2-deoxy-2-(2-oxo-1,2-dihydro-3-(pyrrol-2-yl)-4-methyl-quinolin-7-yl)amino-L-glucose,    2-deoxy-2-(2-oxo-1,2-dihydro-3-phenyl-4-trifluoromethyl-quinolin-7-yl)amino-L-glucose,    2-deoxy-2-(2-oxo-1,2-dihydro-3-(pyridin-2-yl)-4-trifluoromethyl-quinolin-7-yl)amino-L-glucose,    2-deoxy-2-(2-oxo-1,2-dihydro-3-(pyridin-4-yl)-4-trifluoromethyl-quinolin-7-yl)amino-L-glucose,    2-deoxy-2-(2-oxo-1,2-dihydro-3-(thiophen-2-yl)-4-trifluoromethyl-quinolin-7-yl)amino-L-glucose,    2-deoxy-2-(2-oxo-1,2-dihydro-3-(furan-2-yl)-4-trifluoromethyl-quinolin-7-yl)amino-L-glucose,    2-deoxy-2-(2-oxo-1,2-dihydro-3-(pyrrol-2-yl)-4-trifluoromethyl-quinolin-7-yl)amino-L-glucose,    2-deoxy-2-(2-oxo-1,2-dihydro-3-iodo-quinolin-7-yl)amino-L-glucose,    2-deoxy-2-(2-oxo-1,2-dihydro-4-iodo-quinolin-7-yl)amino-L-glucose,    2-deoxy-2-(2-oxo-1,2-dihydro-3-formyl-quinolin-7-yl)amino-L-glucose,    2-deoxy-2-(2-oxo-1,2-dihydro-4-formyl-quinolin-7-yl)amino-L-glucose,    2-deoxy-2-(2-oxo-1,2-dihydro-3-ethenyl-quinolin-7-yl)amino-L-glucose,    2-deoxy-2-(2-oxo-1,2-dihydro-4-ethenyl-quinolin-7-yl)amino-L-glucose,    2-deoxy-2-(2-oxo-1,2-dihydro-3-ethynyl-quinolin-7-yl)amino-L-glucose,    2-deoxy-2-(2-oxo-1,2-dihydro-4-ethynyl-quinolin-7-yl)amino-L-glucose,    2-deoxy-2-(2-oxo-1,2-dihydro-3,4-dimethyl-quinolin-7-yl)amino-L-glucose,    2-deoxy-2-(2-oxo-1,2-dihydro-3-acetyl-quinolin-7-yl)amino-L-glucose,    2-deoxy-2-(2-oxo-1,2-dihydro-4-acetyl-quinolin-7-yl)amino-L-glucose,    2-deoxy-2-(2-oxo-1,2-dihydro-3-cyclopropanyl-quinolin-7-yl)amino-L-glucose,    2-deoxy-2-(2-oxo-1,2-dihydro-4-cyclopropanyl-quinolin-7-yl)amino-L-glucose,    2-deoxy-2-(2-oxo-1,2-dihydro-3-acetyl-4-methyl-quinolin-7-yl)amino-L-glucose,    2-deoxy-2-(2-oxo-1,2-dihydro-3-methyl-4-acetyl-quinolin-7-yl)amino-L-glucose,    2-deoxy-2-(2-oxo-1,2-dihydro-3-propenyl-quinolin-7-yl)amino-D-glucose,    2-deoxy-2-(2-oxo-1,2-dihydro-4-propenyl-quinolin-7-yl)amino-L-glucose,    2-deoxy-2-(1-methyl-2-oxo-1,2-dihydro-quinolin-7-yl)amino-L-glucose,    and 2-deoxy-2-(2-methoxyquinolin-7-yl)amino-L-glucose.    9. The glucose derivative according to the above item 1, which is    selected from the group consisting of the following compounds:-   2-deoxy-2-(2-oxo-2H-chromen-7-yl)amino-D-glucose (in the present    specification, also referred to as CDG),    2,4-dideoxy-2-(2-oxo-2H-chromen-7-yl)amino-4-fluoro-D-glucose    (4-F-CDG),    2,6-dideoxy-2-(2-oxo-2H-chromen-7-yl)amino-6-fluoro-D-glucose    (6-F-CDG), 2-deoxy-2-(2-oxo-1,2-dihydroquinolin-7-yl)amino-D-glucose    (QDG), 2-deoxy-2-(2-oxo-2H-3-methyl-chromen-7-yl)amino-D-glucose    (3-MCDG), 2-deoxy-2-(2-oxo-2H-4-methyl-chromen-7-yl)amino-D-glucose    (4-MCDG),    2-deoxy-2-(2-oxo-2H-3-trifluoromethyl-chromen-7-yl)amino-D-glucose    (3-TFMCDG),    2-deoxy-2-(2-oxo-2H-4-trifluoromethyl-chromen-7-yl)amino-D-glucose    (4-TFMCDG),    2-deoxy-2-(2-oxo-1,2-dihydro-3-methyl-quinolin-7-yl)amino-D-glucose    (3-MQDG),    2-deoxy-2-(2-oxo-1,2-dihydro-4-methyl-quinolin-7-yl)amino-D-glucose    (4-MQDG),    2-deoxy-2-(2-oxo-1,2-dihydro-3-trifluoromethyl-quinolin-7-yl)amino-D-glucose    (3-TFMQDG),    2-deoxy-2-(2-oxo-1,2-dihydro-4-trifluoromethyl-quinolin-7-yl)amino-D-glucose    (4-TFMQDG), 2-deoxy-2-(2-oxo-2H-chromen-7-yl)amino-L-glucose (CLG),    2,4-dideoxy-2-(2-oxo-2H-chromen-7-yl)amino-4-fluoro-L-glucose    (4-F-CLG),    2,6-dideoxy-2-(2-oxo-2H-chromen-7-yl)amino-6-fluoro-L-glucose    (6-F-CLG), 2-deoxy-2-(2-oxo-1,2-dihydroquinolin-7-yl)amino-L-glucose    (QLG), 2-deoxy-2-(2-oxo-2H-3-methyl-chromen-7-yl)amino-L-glucose    (3-MCLG), 2-deoxy-2-(2-oxo-2H-4-methyl-chromen-7-yl)amino-L-glucose    (4-MCLG),    2-deoxy-2-(2-oxo-2H-3-trifluoromethyl-chromen-7-yl)amino-L-glucose    (3-TFMCLG),    2-deoxy-2-(2-oxo-2H-4-trifluoromethyl-chromen-7-yl)amino-L-glucose    (4-TFMCLG),    2-deoxy-2-(2-oxo-1,2-dihydro-3-methyl-quinolin-7-yl)amino-L-glucose    (3-MQLG),    2-deoxy-2-(2-oxo-1,2-dihydro-4-methyl-quinolin-7-yl)amino-L-glucose    (4-MQLG),    2-deoxy-2-(2-oxo-1,2-dihydro-3-trifluoromethyl-quinolin-7-yl)amino-L-glucose    (3-TFMQLG), and    2-deoxy-2-(2-oxo-1,2-dihydro-4-trifluoromethyl-quinolin-7-yl)amino-L-glucose    (4-TFMQLG).    10. The glucose derivative according to the above item 9, which is    2-deoxy-2-(2-oxo-2H-chromen-7-yl)amino-D-glucose (CDG),    2-deoxy-2-(2-oxo-1,2-dihydroquinolin-7-yl)amino-D-glucose (QDG),    2-deoxy-2-(2-oxo-2H-chromen-7-yl)amino-L-glucose (CLG), or    2-deoxy-2-(2-oxo-1,2-dihydroquinolin-7-yl)amino-L-glucose (QLG).    11. A radiolabeled glucose derivative comprising a glucose    derivative according to any one of the above items 1 to 10, wherein    a hydroxy group or a fluorine group at any one of the 2-, 4-, and    6-positions of the glucose is substituted by ¹⁸F, and which is    preferably    2,4-dideoxy-2-(2-oxo-2H-chromen-7-yl)amino-4-[¹⁸F]fluoro-D-glucose    (4-¹⁸F-CDG),    2,4-dideoxy-2-(2-oxo-2H-chromen-7-yl)amino-4-[¹⁸F]fluoro-L-glucose    (4-¹⁸F-CLG),    2,6-dideoxy-2-(2-oxo-2H-chromen-7-yl)amino-6-[¹⁸F]fluoro-D-glucose    (6-¹⁸F-CDG), or    2,6-dideoxy-2-(2-oxo-2H-chromen-7-yl)amino-6-[¹⁸F]fluoro-L-glucose    (6-¹⁸F-CLG).    12. A radiolabeled glucose derivative comprising a glucose    derivative according to any of the above items 1 to 9, wherein R₁ or    R₂ is ¹¹CH₃ or CF₂ ¹⁸F, and which is preferably    2-deoxy-2-(2-oxo-2H-3-[¹¹C]methyl-chromen-7-yl)amino-D-glucose    (3-[¹¹C]MCDG),    2-deoxy-2-(2-oxo-2H-4-[¹¹C]methyl-chromen-7-yl)amino-D-glucose    (4-[¹¹C]MCDG),    2-deoxy-2-(2-oxo-2H-3-[¹⁸F]fluorodifluoromethyl-chromen-7-yl)amino-D-glucose    (3-[¹⁸F]TFMCDG),    2-deoxy-2-(2-oxo-2H-4-[¹⁸F]fluorodifluoromethyl-chromen-7-yl)amino-D-glucose    (4-[¹⁸F]TFMCDG),    2-deoxy-2-(2-oxo-1,2-dihydro-3-[¹¹C]methyl-quinolin-7-yl)amino-D-glucose    (3-[¹¹C]MQDG),    2-deoxy-2-(2-oxo-1,2-dihydro-4-[¹¹C]methyl-quinolin-7-yl)amino-D-glucose    (4-[¹¹C]MQDG),    2-deoxy-2-(2-oxo-1,2-dihydro-3-[¹⁸F]fluorodifluoromethyl-quinolin-7-yl)amino-D-glucose    (3-[¹⁸F]=QDG),    2-deoxy-2-(2-oxo-1,2-dihydro-4-[¹⁸F]fluorodifluoromethyl-quinolin-7-yl)amino-D-glucose    (4-[¹⁸F]TFMQDG),    2-deoxy-2-(2-oxo-2H-3-[¹¹C]methyl-chromen-7-yl)amino-L-glucose    (3-[¹¹C]MCLG),    2-deoxy-2-(2-oxo-2H-4-[¹¹C]methyl-chromen-7-yl)amino-L-glucose    (4-[¹¹C]MCLG),    2-deoxy-2-(2-oxo-2H-3-[¹⁸F]fluorodifluoromethyl-chromen-7-yl)amino-L-glucose    (3-[¹⁸F]=CLG),    2-deoxy-2-(2-oxo-2H-4-[¹⁸F]fluorodifluoromethyl-chromen-7-yl)amino-L-glucose    (4-[¹⁸F]TFMCLG),    2-deoxy-2-(2-oxo-1,2-dihydro-3-[¹¹C]methyl-quinolin-7-yl)amino-L-glucose    (3-[¹¹C]MQLG),    2-deoxy-2-(2-oxo-1,2-dihydro-4-[¹¹C]methyl-quinolin-7-yl)amino-L-glucose    (4-[¹¹C]MQLG),    2-deoxy-2-(2-oxo-1,2-dihydro-3-[¹⁸F]fluorodifluoromethyl-quinolin-7-yl)amino-L-glucose    (3-[¹⁸F]TFMQLG), or    2-deoxy-2-(2-oxo-1,2-dihydro-4-[¹⁸F]fluorodifluoromethyl-quinolin-7-yl)amino-L-glucose    (4-[¹⁸F]TFMQLG).    13. A composition for imaging a target cell, comprising a glucose    derivative according to any one of the above items 1 to 12.    14. A composition for imaging a target cell, comprising a glucose    derivative according to the above items 3 or 4.    15. A composition for imaging a target cell, comprising a glucose    derivative according to the above items 6 or 7.    16. A composition for imaging a target cell, comprising a glucose    derivative according to the above item 9.    17. A composition for imaging a target cell, comprising a glucose    derivative according to the above item 10.    18. The composition according to the above item 17, wherein the    glucose derivative is    2-deoxy-2-(2-oxo-2H-chromen-7-yl)amino-D-glucose (CDG) or    2-deoxy-2-(2-oxo-2H-chromen-7-yl)amino-L-glucose (CLG).    19. The composition according to the above item 17, wherein the    glucose derivative is    2-deoxy-2-(2-oxo-1,2-dihydroquinolin-7-yl)amino-D-glucose (QDG) or    2-deoxy-2-(2-oxo-1,2-dihydroquinolin-7-yl)amino-L-glucose (QLG).    20. A composition for imaging a target cell, comprising a    radiolabeled glucose derivative according to the above item 11 or    12.    21. A method for imaging a target cell, comprising the following    steps:    a. contacting a glucose derivative according to any one of the above    items 1 to 12 with the target cell; and    b. detecting fluorescence emitted by the glucose derivative present    within the target cell.    22. The imaging method according to the above item 21, wherein the    glucose derivative is    2-deoxy-2-(2-oxo-2H-chromen-7-yl)amino-D-glucose (CDG) or    2-deoxy-2-(2-oxo-2H-chromen-7-yl)amino-L-glucose (CLG).    23. The imaging method according to the above item 21, wherein the    glucose derivative is    2-deoxy-2-(2-oxo-1,2-dihydroquinolin-7-yl)amino-D-glucose (QDG) or    2-deoxy-2-(2-oxo-1,2-dihydroquinolin-7-yl)amino-L-glucose (QLG).    24. The imaging method according to any one of the above items 21 to    23, wherein the composition in the step a further comprises an    additional fluorescently labeled glucose derivative, and the step b    is the step of detecting at least one of the glucose derivatives    present within the target cell.    25. The imaging method according to the above item 24, wherein the    additional fluorescently labeled glucose derivative is at least one    glucose derivative selected from the group consisting of    2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-D-glucose    (2-NBDG),    2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-L-glucose    (2-NBDLG),    2-deoxy-2-[N-7-(N′,N′-dimethylaminosulfonyl)benz-2-oxa-1,3-diazol-4-yl)amino]-D-glucose    (2-DBDG),    2-deoxy-2-[N-7-(N′,N′-dimethylaminosulfonyl)benz-2-oxa-1,3-diazol-4-yl)amino]-L-glucose    (2-DBDLG), and 2-amino-2-deoxy-L-glucose having a sulforhodamine    (preferably sulforhodamine 101 or sulforhodamine B) bound to the    2-position through a sulfonamide bond.    26. The imaging method according to the above item 25, wherein the    combination of the glucose derivatives is a combination of two    glucose derivatives that respectively emit two colors selected from    blue, green, and red colors as fluorescence.    27. The imaging method according to the above item 25, wherein the    combination of the glucose derivatives is a combination of a glucose    derivative that emits blue color as fluorescence, a glucose    derivative that emits green color as fluorescence, and a glucose    derivative that emits red color as fluorescence.    28. A method for detecting a cancer or a cancer cell, comprising the    following steps:    a. contacting a composition containing a glucose derivative    according to the above item 3 or 4 with a target cell; and    b. detecting the glucose derivative present within the target cell.    29. The detection method according to the above item 28, wherein the    glucose derivative is CDG, QDG, 3-TFMCDG, 4-TFMCDG, 3-TFMQDG, or    4-TFMQDG.    30. The detection method according to the above item 29, wherein the    glucose derivative is CDG.    31. A method for detecting a cancer or a cancer cell, comprising the    following steps:    a. contacting a composition containing a glucose derivative    according to the above item 6 or 7 with a target cell; and    b. detecting the glucose derivative present within the target cell.    32. The detection method according to the above item 31, wherein the    glucose derivative is CLG, QLG, 3-TFMCLG, 4-TFMCLG, 3-TFMQLG, or    4-TFMQLG.    33. The detection method according to the above item 32, wherein the    glucose derivative is CLG.    34. The detection method according to any one of the above items 28    to 33, wherein the detection in the step b is performed by imaging    the target cell.    35. The detection method according to any one of the above items 28    to 34, wherein the composition in the step a further comprises an    additional fluorescently labeled glucose derivative, and the step b    is the step of detecting at least one of the glucose derivatives    present within the target cell.    36. The detection method according to the above item 35, wherein the    additional fluorescently labeled glucose derivative is at least one    glucose derivative selected from the group consisting of    2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-D-glucose    (2-NBDG),    2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-L-glucose    (2-NBDLG),    2-deoxy-2-[N-7-(N′,N′-dimethylaminosulfonyl)benz-2-oxa-1,3-diazol-4-yl)amino]-D-glucose    (2-DBDG),    2-deoxy-2-[N-7-(N′,N′-dimethylaminosulfonyl)benz-2-oxa-1,3-diazol-4-yl)amino]-L-glucose    (2-DBDLG), and 2-amino-2-deoxy-L-glucose having a sulforhodamine    (preferably sulforhodamine 101 or sulforhodamine B) bound to the    2-position through a sulfonamide bond.    37. The detection method according to the above item 36, wherein the    combination of the glucose derivatives is a combination of two    glucose derivatives that respectively emit two colors selected from    blue, green, and red colors as fluorescence.    38. The detection method according to the above item 36, wherein the    combination of the glucose derivatives is a combination of a glucose    derivative that emits blue color as fluorescence, a glucose    derivative that emits green color as fluorescence, and a glucose    derivative that emits red color as fluorescence.    39. An imaging agent for imaging a target cancer cell, comprising a    glucose derivative according to the above item 3 or 4.    40. The imaging agent according to the above item 39, wherein the    glucose derivative is CDG, QDG, 4-F-CDG, 6-F-CDG, 4-F-QDG, 6-F-QDG,    3-TFMCDG, 4-TFMCDG, 3-TFMQDG, or 4-TFMQDG.    41. The imaging agent according to the above item 40, wherein the    glucose derivative is CDG.    42. An imaging agent for imaging a target cancer cell, comprising a    glucose derivative according to the above item 6 or 7.    43. The imaging agent according to the above item 42, wherein the    glucose derivative is CLG, QLG, 4-F-CLG, 6-F-CLG, 4-F-QLG, 6-F-QLG,    3-TFMCLG, 4-TFMCLG, 3-TFMQLG, or 4-TFMQLG.    44. The imaging agent according to the above item 43, wherein the    glucose derivative is CLG.    45. An imaging agent for PET examination of a cancer, comprising a    radiolabeled glucose derivative according to the above item 11 or    12.

The present invention provides a novel glucose derivative, which istaken into cells via a membrane sugar transport system, and furtherprovides an imaging method and an imaging agent capable of identifyingcells or intracellular molecules with high contrast. The presentinvention also provides a method capable of identifying cancer cellswith high contrast, and an imaging agent therefor.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows the comparison of the fluorescence spectra of CDG, QDG, and2-NBDLG.

FIG. 2 shows results of confirming the uptake of CDG (100 μM) into mouseinsulinoma cells (MIN6), and the influence of a glucose transportinhibitor on the uptake. FIG. 2A shows the results of confirming theuptake of CDG and the influence of an inhibitor cytochalasin B (CB).FIG. 2B shows the results of confirming the uptake of CDG and theinfluence of an inhibitor phloretin (PHT). FIG. 2C shows the results ofconfirming the uptake of CDG or CLG and the influence of an inhibitorphloretin (PHT).

FIG. 3 shows results of examining the uptake of a green fluorescentD-glucose derivative 2-NBDG into mouse insulinoma cells (MIN6), and theinhibitory effect of CB or PHT on the uptake.

FIG. 4 shows results of confirming the uptake of CDG or CLG into mouseinsulinoma cells (MIN6), and the influence of excessive D-glucose orL-glucose thereon.

FIG. 5 shows results of confirming the uptake of QDG (100 μM) into mouseinsulinoma cells (MIN6), and the influence of a glucose transportinhibitor on the uptake. FIG. 5A shows the results of confirming theinfluence of cytochalasin B (CB). FIG. 5B shows the results ofconfirming the influence of phloretin (PHT).

FIG. 6 shows fluorescence microscopic images which were taken 6 minutesafter the start of washout of a CDG+2-NBDLG+2-TRLG mixed solution whichhad been administered for 3-minute to MIN6 cell masses that formedspheroids. FIGS. 6A, 6B, and 6C show the uptake into cells of bluefluorescence-emitting CDG, green fluorescence-emitting 2-NBDLG, and redfluorescence-emitting 2-TRLG, respectively. FIG. 6D is a differentialinterference contrast (DIC) image.

FIG. 7 is a superimposed image of FIGS. 6A and 6B. However, the uptakeof CDG is expressed in red color instead of blue color in order tofacilitate visualizing the localization of CDG and 2-NBDLG.

FIG. 8 shows fluorescence microscopic images which were taken 6 minutesafter the start of washout of a CLG+2-NBDLG+2-TRLG mixed solution whichhad been administered for 3-minute to MIN6 cell masses that formedspheroids. FIGS. 8A, 8B, and 8C show the uptake into cells of bluefluorescence-emitting CLG, green fluorescence-emitting 2-NBDLG, and redfluorescence-emitting 2-TRLG, respectively. FIG. 8D is a differentialinterference contrast (DIC) image.

FIG. 9 shows fluorescence emitted by cells which was observed in a bluefluorescence wavelength region suitable for the observation of CLGbefore administration of a CLG+2-TRLG mixed solution to acutely isolatednormal neuronal cells and 8 minutes after the start of washout following3-minute administration of the mixed solution. FIG. 9A is anautofluorescence image taken before the administration of the mixedsolution. The arrows indicate two normal neuronal cells, and * indicatesthe debris of killed cells. FIG. 9B shows a differential interferencecontrast (DIC) image superimposed on the image of FIG. 9A in order tofacilitate visualizing the positions of the cells. FIG. 9C is a bluefluorescence image taken 8 minutes after the start of washout of themixed solution. FIG. 9D is a DIC image superimposed on the image of FIG.9C.

FIG. 10 shows fluorescence microscopic images showing the change influorescence intensity exhibited by the cells between before and afterthe administration of the CLG+2-TRLG mixed solution in FIG. 9, asobserved in a red fluorescence wavelength region suitable for theobservation of 2-TRLG. FIG. 10A shows autofluorescence before theadministration of the mixed solution. FIG. 10B shows a differentialinterference contrast (DIC) image superimposed on the image of FIG. 10Ain order to facilitate visualizing the positions of cells. FIG. 10C is ared fluorescence image taken 8 minutes after the start of washout of themixed solution. FIG. 10D is a DIC image superimposed on the image ofFIG. 10C. Intense red color at the central portion of each imageresulting from the leakage of a portion of irradiation light to thedetector side through a fluorescence filter since weak red fluorescencewas photographed with sensitivity enhanced.

FIG. 11 shows results of change in fluorescence intensity exhibited byacutely isolated normal neuronal cells (arrow) between before and after3-minute administration of a CDG+2-TRLG mixed solution to the cells, asobserved in the wavelength region (blue channel) of blue fluorescenceemitted by CDG.

FIG. 12 shows results of change in fluorescence between before and afterthe administration of the mixed solution in FIG. 11, as simultaneouslyobserved in the wavelength region (red channel) of red fluorescenceemitted by 2-TRLG.

DESCRIPTION OF EMBODIMENTS

In the present specification, the “target cell” is used in the meaningincluding the targeted cell itself and various organs or moleculespresent within the cell. Thus, the phrase “imaging a (target) cell”described herein is used in the meaning including imaging any one ormore of the targeted cell itself and various organs or molecules presentwithin the cell, etc.

Examples of the subject to be imaged can include such targets as cellmembranes and/or intracellular regions called cytosol, subcellular smallorgans (so-called organelles examples of which can include the inside ofstructures such as nucleus, endoplasmic reticulum, Golgi body, endosome,lysosome, mitochondrion, peroxisome, autophagosome, glycosome,proteasome, vacuole, chloroplast, and glyoxysome, and/or biomembranessurrounding these structures), structures in the inside and/or on thesurface of the organelles (examples of which can include nucleolus andribosome), and molecules within the target cell (e.g., molecules presentin the inside of the cell, i.e., within cytoplasm or nucleus, moleculespresent in the cell membrane of the target cell, and molecules presenton the cell membrane of the target cell). The glucose derivative of thepresent invention can be used for imaging at least one of thesesubjects. The subject to be imaged is preferably a cell membrane and/oran intracellular region called cytosol, a subcellular small organ, or astructure in the inside and/or on the surface of subcellular organelles,particularly preferably cytosol or nucleus.

(I) Glucose Derivative

The glucose derivative of the present invention is a compoundrepresented by the following formula (1):

or a salt thereof.

In the formula (1), X—Y—Z represents O—C═O, NH—C═O, NR₃—C═O, or N═C—OR₄,wherein R₃ represents C₁-C₅ alkyl, and R₄ represents C₁-C₅ alkyl.

R₁ and R₂ are each independently selected from the group consisting ofhydrogen, halogen, C₁-C₅ alkyl, C₂-C₅ alkenyl, C₂-C₅ alkynyl, C₁-C₅haloalkyl, C₁-C₅ alkylamino, cycloalkyl, phenyl, pyridyl, thiophenyl,pyrrolyl, and furanyl.

By virtue of the above a structure, the glucose derivative of thepresent invention has a fluorophore within its molecule.

In the formula (1), G represents D-amino-glucose or L-amino-glucose. Theglucose derivative of the present invention has a fluorophore via anamino group at the 2- or 6-position of glucose and preferably has afluorophore via the amino group at the 2-position. Specifically, G isany of the structures of the following formulas (G1) to (G4):

In these formulas, Ra and Rb each represent hydroxy or fluorine. Whenone of Ra and Rb is fluorine, the other group is hydroxy. Preferably,both of Ra and Rb are hydroxy.

In the formula (1) which represents the glucose derivative of thepresent invention, X—Y—Z is O—C═O, NH—C═O, NR₃—C═O, or N═C—OR₄ (whereinR₃ is C₁-C₅ alkyl, and R₄ is C₁-C₅ alkyl), preferably O—C═O, NH—C═O, orN(CH₃)—C═O, particularly preferably O—C═O or NH—C═O.

In the formula (1) which represents the glucose derivative of thepresent invention, R₁ and R₂ are each independently selected from thegroup consisting of hydrogen, halogen, C₁-C₅ alkyl, C₂-C₅ alkenyl, C₂-C₅alkynyl, C₁-C₅ haloalkyl, C₁-C₅ alkylamino, cycloalkyl, phenyl, pyridyl,thiophenyl, pyrrolyl, and furanyl, and, preferably, R₁ and R₂ are eachindependently hydrogen, halogen, C₁-C₅ alkyl, C₂-C₅ alkenyl, C₂-C₅alkynyl, or C₁-C₅ haloalkyl, more preferably, R₁ and R₂ are eachindependently hydrogen, C₁-C₅ alkyl, or C₁-C₅ haloalkyl, furtherpreferably, R₁ and R₂ are each independently hydrogen, methyl, orfluoromethyl, and particularly preferably, R₁ and R₂ are hydrogen.

A feature of the glucose derivative of the present invention is that thefluorophore is directly bound to the 2- or 6-position of glucose via NH.As a result, the glucose derivative of the present invention is takeninto cells via a membrane sugar transport system such as GLUT.

A preferred form of the glucose derivative of the present invention isany of the following compounds:

2-Deoxy-2-(2-oxo-2H-chromen-7-yl)amino-D-glucose (referred to as CDG)(D-glucose derivative) and2-deoxy-2-(2-oxo-2H-chromen-7-yl)amino-L-glucose (referred to as CLG)(L-glucose derivative) are in the relationship of enantiomers. Theirmaximum excitation wavelength (Ex max) and maximum fluorescencewavelength (Em max) are 366.5 nm (Ex max) and 454.5 nm (Em max).

Another preferred form of the glucose derivative of the presentinvention is any of the following compounds:

2-Deoxy-2-(2-oxo-1,2-dihydroquinolin-7-yl)amino-D-glucose (referred toas QDG) (D-glucose derivative) and2-deoxy-2-(2-oxo-1,2-dihydroquinolin-7-yl)amino-L-glucose (referred toas QLG) (L-glucose derivative) are in the relationship of enantiomers.Their maximum excitation wavelength (Ex max) and maximum fluorescencewavelength (Em max) are 353.5 nm (Ex max) and 423.0 nm (Em max).

Further examples of the preferred form of the glucose derivative of thepresent invention can include compounds having a substituent at the 3-or 4-position of the coumarin backbone or quinoline backbone in CDG,CLG, QDG, or QLG described above. Examples of the substituent caninclude halogen, acetyl, formyl, allyl, ethynyl, propenyl, methyl,fluoromethyl, cycloalkyl, phenyl, pyridyl, thiophenyl, pyrrolyl, andfuranyl. The substituent is particularly preferably methyl orfluoromethyl.

Examples of the preferred form of the glucose derivative of the presentinvention can include the following compounds.

TABLE 1 Compound No. Structure Compound 101 CDG

Compound 102

Compound 103

Compound 104

Compound 105

Compound 106

Compound 107

Compound 108

Compound 201 CLG

Compound 202

Compound 203

Compound 204

Compound 205

Compound 206

Compound 207

Compound 208

TABLE 2 Compound 109

Compound 110

Compound 111

Compound 112

Compound 113

Compound 114

Compound 115

Compound 209

Compound 210

Compound 211

Compound 212

Compound 213

Compound 214

Compound 215

TABLE 3 Compound 116

Compound 117

Compound 118

Compound 119

Compound 120

Compound 121

Compound 122

Compound 216

Compound 217

Compound 218

Compound 219

Compound 220

Compound 221

Compound 222

TABLE 4 Compound 123

Compound 124 QDG

Compound 125

Compound 126

Compound 127

Compound 128

Compound 129

Compound 130

Compound 223

Compound 224 QLG

Compound 225

Compound 226

Compound 227

Compound 228

Compound 229

Compound 230

TABLE 5 Compound 131

Compound 132

Compound 133

Compound 134

Compound 135

Compound 136

Compound 137

Compound 231

Compound 232

Compound 233

Compound 234

Compound 235

Compound 236

Compound 237

TABLE 6 Compound 138

Compound 139

Compound 140

Compound 141

Compound 142

Compound 143

Compound 144

Compound 238

Compound 239

Compound 240

Compound 241

Compound 242

Compound 243

Compound 244

TABLE 7 Compound 145

Compound 146

Compound 147

Compound 148

Compound 149

Compound 150

Compound 151

Compound 152

Compound 245

Compound 246

Compound 247

Compound 248

Compound 249

Compound 250

Compound 251

Compound 252

TABLE 8 Compound 153

Compound 154

Compound 155

Compound 156

Compound 157

Compound 158

Compound 159

Compound 160

Compound 253

Compound 254

Compound 255

Compound 256

Compound 257

Compound 258

Compound 259

Compound 260

TABLE 9 Compound 161

Compound 162

Compound 163

Compound 164

Compound 165

Compound 166

Compound 167

Compound 261

Compound 262

Compound 263

Compound 264

Compound 265

Compound 266

Compound 267

TABLE 10 Compound 168

Compound 169

Compound 170

Compound 171

Compound 172

Compound 173

Compound 174

Compound 175

Compound 268

Compound 269

Compound 270

Compound 271

Compound 272

Compound 273

Compound 274

Compound 275

TABLE 11 Compound 176

Compound 177

Compound 178

Compound 179

Compound 180

Compound 181

Compound 182

Compound 276

Compound 277

Compound 278

Compound 279

Compound 280

Compound 281

Compound 282

The glucose derivative of the present invention is particularlypreferably selected from CDG, CLG, QDG, and QLG.

The glucose derivative of the present invention can be used after beingdissolved in an arbitrary solution, for example, a solvent such as wateror aqueous dimethyl sulfoxide. Also, the glucose derivative of thepresent invention is stable even in a solvent or solution used for theimaging of cells or intracellular molecules, particularly, in a buffersolution, and as such, is suitable as an imaging agent.

Hereinafter, a method for synthesizing the D-glucose derivative of thepresent invention will be mainly described. The L-glucose derivative ofthe present invention can be synthesized by using an L-form of sugar asa starting material.

The binding position of the fluorescent molecular group having acoumarin backbone or a quinoline backbone to glucose is either the 2- or6-position of glucose, preferably the 2-position. The 2-substitutedderivative can be synthesized using glucosamine, and the 6-substitutedderivative can be synthesized using a 6-deoxy-6-amino-glucosederivative.

D-Glucosamine or L-glucosamine can be used as the glucosamine.Synthesized D-glucosamine or commercially available D-glucosamine can beused as the D-glucosamine. The L-glucosamine can be synthesized by amethod described in WO2010/16587 (Patent Literature 2) or a methoddescribed in WO2012/133688 (Patent Literature 4) (the description ofthese bulletins or publications of the applications are incorporatedherein by reference). The method for synthesizing L-glucosaminedescribed in WO2012/133688 is as follows:

The 6-deoxy-6-amino-glucose derivative can be synthesized, for example,through 7 stages from glucose as follows, though the synthesis method isnot limited thereto. The 6-deoxy-6-amino-glucose derivative can besynthesized by converting the hydroxy group at the 1-position of glucoseto methyl glycoside, protecting the 4- and 6-positions with benzylidenegroups and the 2- and 3-positions with isopropylidene groups,deprotecting the benzylidene groups, converting the 6-position to atoluenesulfonyl group and then to an azide group, and reducing the azidegroup to an amino group. Commercially available D-glucose orcommercially available L-glucose can be used as the glucose.

The D-glucose derivative of the present invention can be synthesized,for example, as follows, though the synthesis method is not limitedthereto.

Compound (A) is synthesized which is a compound having, as afluorophore, a coumarin backbone substituted at the 7-position with anactive group. The compound (A) is synthesized by binding atrifluoromethanesulfonyl group to the hydroxy group at the 7-position.

Compound (B) is synthesized which is a compound having, a fluorophore, aquinolinone backbone substituted at the 7-position with an active group.This compound is synthesized through 2 stages from an aniline derivativesubstituted at the 3-position with iodine.

Aside therefrom, D-glucosamine having protected OH at the 1-, 4-, and6-positions is synthesized, for example, as follows:

Subsequently, a synthesis can be performed by condensing the compound Aand the D-glucosamine having protected OH at the 1- and 6-positions, forexample, by C—N cross-coupling reaction using palladium, so as to bondthe compound A to D-glucose via —NH—

The D-glucose derivative of the present invention having a coumarinbackbone or quinoline backbone at the 6-position of glucose can besynthesized by condensing the compound A and the6-deoxy-6-amino-D-glucose derivative having protected OH at the 1-, 2-,and 3-positions, for example, by C—N cross-coupling reaction usingpalladium, so as to bond the compound A to D-glucose via —NH—.

The L-glucose derivative of the present invention can be synthesized inthe same way as above by using L-glucosamine instead of D-glucosamine.

The glucose derivative having a coumarin backbone or quinoline backbonehaving a substituent at the 3- or 4-position can be synthesized, forexample, by a Suzuki coupling method using CDG, CLG, QDG, or QLGsynthesized according to a method disclosed herein, or by synthesizing acompound (A) having a coumarin backbone or quinoline backbone having asubstituent at the 3- or 4-position and then reacting the compound (A)with glucosamine by use of a direct introduction method by C—Hactivation, though the synthesis method is not limited thereto.

Hereinafter, a method for synthesizing a D-glucose derivative which hasa coumarin with an aromatic group bound at the 3-position thereof willbe illustrated.

(i) Suzuki Coupling Method:

(ii) Direct Introduction Method by C—H Activation:

A radiolabel can also be added to the glucose derivative of the presentinvention. As a result, imaging based on radioactivity, for example, PETimaging, can also be achieved. In addition, such a compound also permitsimaging in a dual mode using fluorescence and radiation.

Examples of the method for adding a radiolabel to the glucose derivativeof the present invention can include a method of substituting a hydroxygroup of glucose by ¹⁸F, and a method of radiolabeling a substituent inthe coumarin backbone or quinoline backbone.

In the former case, the position of the hydroxy group to be substitutedby ¹⁸F is the 4- or 6-position when the coumarin backbone or quinolinebackbone resides at the 2-position of glucose, or is the 2- or4-position when the coumarin backbone or quinoline backbone resides atthe 6-position of glucose. Examples of the resulting glucose derivativecan include 4-¹⁸F-CDG, 4-¹⁸F-CLG, 6-¹⁸F-CDG, 6-¹⁸F-CLG, 4-¹⁸F-QDG,4-¹⁸F-QLG, 6-¹⁸F-QDG, and 6-¹⁸F-QLG.

In the latter case, examples of the radiolabeled glucose derivative caninclude a compound in which the carbon atom of the methyl group bound tothe 3- or 4-position of the coumarin backbone or quinoline backbone issubstituted by ¹¹C, and a compound in which one of the fluorine atoms ofthe trifluoromethyl group bound to the 3- or 4-position of the coumarinbackbone or quinoline backbone is substituted by ¹⁸F. Examples thereofcan include 3-[¹¹C]MCDG, 4-[¹¹C]MCDG, 3-[¹⁸F]TFMCDG, 4-[¹⁸F]TFMCDG,3-[¹¹C]MQDG, 4-[¹¹C]MQDG, 3-[¹⁸F]TFMQDG, 4-[¹⁸F]TFMQDG, 3-[¹¹C]MCLG,4-[¹¹C]MCLG, 3-[¹⁸F]TFMCLG, 4-[¹⁸F]TFMCLG, 3-[¹¹C]MQLG, 4-[¹¹C]MQLG,3-[¹⁸F]TFMQLG, and 4-[¹⁸F]TFMQLG.

Such a radiolabeled glucose derivative can be synthesized, for example,as follows, though the synthesis method is not limited thereto.

(i) Method for Synthesizing a Compound Containing a Radioisotope ¹⁸F at6-Position of Glucose

For example, the following compound (6-¹⁸F-CLG) can be synthesized, forexample, by steps given below.

(ii) Method for Synthesizing a Compound Containing a Radioisotope ¹⁸F at4-Position of Glucose

For example, the following compound (4-¹⁸F-CLG) can be synthesized, forexample, by steps given below.

(iii) Method for Synthesizing a Compound Containing a Radioisotope ¹⁸Fat 4-Position of Glucose

For example, the following compound (4-¹⁸F-6-CDG) can be synthesized,for example, by steps given below.

(iv) Method for Synthesizing a Compound Containing a Radioisotope ¹⁸F at2-Position of Glucose

For example, the following compound (2-¹⁸F-6-CDG) can be synthesized,for example, by steps given below.

(v) Method for Synthesizing a Derivative Containing a Radioisotope ¹⁸Fin a Coumarin Backbone—(1)

For example, the following compound (¹⁸F-3-[¹⁸F]TFMCDG) can besynthesized, for example, by steps given below.

A derivative containing a radioisotope ¹⁸F in a quinoline backbone canbe synthesized in the same way as above.

(vi) Method for Synthesizing a Derivative Containing a Radioisotope ¹⁸Fin a Coumarin Backbone—(2)

For example, the following compound (¹⁸F-4-[¹⁸F]TFMCDG) can besynthesized, for example, by steps given below.

A derivative containing a radioisotope ¹⁸F in a quinoline backbone canbe synthesized in the same way as above.

(vii) Method (1) for Synthesizing a Derivative Containing a Radioisotope¹¹C in a Coumarin Backbone

For example, the following compound (¹¹C-3-[¹¹C]MCDG) can besynthesized, for example, by steps given below.

A derivative containing a radioisotope ¹¹C in a quinoline backbone canbe synthesized in the same way as above.

(viii) Method (2) for Synthesizing a Derivative Containing aRadioisotope ¹¹C in a Coumarin Backbone

For example, the following compound (¹¹C-4-[¹¹C]MCDG) can besynthesized, for example, by steps given below.

A derivative containing a radioisotope ¹¹C in a quinoline backbone canbe synthesized in the same way as above.

The fluorophore contained in the glucose derivative of the presentinvention is a fluorophore having a coumarin backbone or quinolinebackbone, that is, a fluorophore that emits blue fluorescence.Therefore, this fluorophore generally has a smaller molecular size thana molecule having a fluorescence maximum in the green, red, ornear-infrared region and as such, is considered to have a fluorescentgroup smaller in steric hindrance against the passage through a membranesugar transport system such as GLUT. In addition, the glucose derivativeof the present invention is featured in that the coumarin backbone orquinoline backbone constituting the fluorophore is directly bound to theglucose backbone via NH. This is considered to provide an advantage thatsteric hindrance against the passage through a membrane sugar transportsystem such as GLUT is reduced.

As a result of conducting pharmacological tests applied to cells, thepresent inventors have confirmed that the aforementioned bluefluorescent D-glucose derivatives designated as CDG and QDG are takeninto cancer cells via GLUT. The present inventors have further developedblue fluorescent L-glucose derivative molecules CLG and QLG, which areenantiomers of CDG and QDG, as control compounds. These four compounds,CDG, CLG, QDG, and QLG, are the first group of blue glucose derivativesthat have been quantitatively shown to have a stereoselectivity betweenD/L-glucose for cellular uptake so as to allow the D form to be takeninto cells via GLUT.

The D-glucose derivative (e.g., CDG or QDG) serving as the glucosederivative of the present invention can emit blue fluorescence as itscontrol compound L-glucose derivative (e.g., CLG or QLG) does.Therefore, the glucose derivative of the present invention is usefulequivalently to or more than the green D-glucose derivative 2-NBDG andthe green L-glucose derivative 2-NBDLG previously reported by thepresent inventors (Patent Literature 2) for elucidating the glucoseuptake mechanism of cells, and can also be used in combinationtherewith.

Specifically, for example, CDG or QDG can be used in combination with2-NBDLG, or CLG or QLG can be used in combination with 2-NBDG, for theelucidation of the stereoselective transport mechanism of glucose insuch a way that the occurrence of GLUT-mediated D-glucose-like cellularuptake and GLUT-free cellular uptake can be compared at the same time inthe same cell selected from various cells, while such combined use canalso offer valuable information for understanding the influence of thefluorescent group on the transport mechanism, though how to use theglucose derivative of the present invention is not limited thereto.

Further, when the blue D-glucose derivative CDG or QDG and the blueL-glucose derivative CLG or QLG of the present invention are examinedfor uptake into cancer cells, D form-dominant uptake was shown, which issimilar to the relationship between the green D-glucose derivative2-NBDG and the green L-glucose derivative 2-NBDLG. Furthermore, the blueL-glucose derivative CLG of the present invention was not taken intonormal cells. Specifically, cancer cells can also be detected using theblue L-glucose derivative of the present invention, for example, CLG orQLG, like the detection of cancer cells using 2-NBDLG previouslyreported by the present inventors (Patent Literature 4).

One aspect of the present invention is a method for imaging a cell or anintracellular molecule using the glucose derivative of the presentinvention.

Another aspect of the present invention is an imaging agent for imaginga cell or an intracellular molecule, comprising the glucose derivativeof the present invention.

The glucose derivative of the present invention is taken into cells viathe membrane sugar transport system of the cells and therefore, can notonly image the cells but can image intracellular molecules and organs,for example, cytoplasm and nucleus.

Yet another aspect of the present invention is an imaging agent fordetecting a cancer cell using the glucose derivative of the presentinvention, preferably the L-glucose derivative, particularly preferablyCLG or QLG.

An alternative aspect of the present invention is a method for detectinga cancer cell using a composition comprising the glucose derivative ofthe present invention, preferably the L-glucose derivative, particularlypreferably CLG or QLG.

A further alternative aspect of the present invention is a PET imagingagent for detecting a cancer cell using a radiolabeled glucosederivative, which is one form of the glucose derivative of the presentinvention, preferably a radiolabeled L-glucose derivative, particularlypreferably radiolabeled 4-F-CLG, 6-F-CLG, TFMCLG, or TFMQLG.

According to the present invention, a target cell can be imaged at anindividual cell level by contacting a composition comprising the glucosederivative of the present invention (hereinafter, also referred to asthe “composition of the present invention” or the “imaging agent of thepresent invention”) as a reagent with the target cell. According to thepresent invention, a cell in a tissue can also be imaged at anindividual cell level by contacting the composition of the presentinvention with the tissue containing the target cell for imaging.

The glucose derivative of the present invention includes both D-glucosederivatives and L-glucose derivatives. By using the D and L forms, thetarget can be imaged at a cell level depending upon the DL configurationof glucose, thereby making it possible to not only elucidate thefunctions of the target but discriminate between normal cells andabnormal cells.

As for microbes having properties different from mammalian cells interms of the recognition, transport, and metabolism of glucoseassociated with the configurations of D and L forms, their functions canalso be elucidated by imaging at a cell level using the D- or L-glucosederivative of the present invention.

According to the present invention, whether or not a target cell is acancer cell can be further determined by contacting a compositioncomprising the glucose derivative of the present invention, preferablythe L-glucose derivative (hereinafter, also referred to as the“composition of the present invention” or the “imaging agent of thepresent invention”) as a reagent with the target cell.

According to the present invention, a cancer cell in a tissue can bedetected by contacting the composition of the present invention with thetissue containing the target cell for imaging.

According to the present invention, cancer cells or a tissue containingthese cells can be detected by administering the composition of thepresent invention to a living body for imaging. This method is useful asa method for detecting a cancer.

Cancer imaging can be achieved by PET using a composition comprising theradiolabeled glucose derivative of the present invention, preferably theradiolabeled L-glucose derivative of the present invention. Examples ofthe radiolabeled glucose derivative of the present invention can include4-F-CLG, 6-F-CLG, 4-F-QLG, 6-F-QLG, TFMCDG, TMFCLG, TMFQDG, and TMFQLGand can preferably include 4-F-CLG, 4-F-QLG, TMFCLG, and TMFQLG.

The composition of the present invention includes compositions,comprising the glucose derivative of the present invention, in any formapplicable to cells, and may be in any form of a solution, a gel andothers without particular limitations as long as the composition isapplicable to cells. The composition can contain any component withoutparticular limitations as long as the component is suitable forapplication to cells. For example, the glucose derivative of the presentinvention may be dissolved in a buffer solution or a medium for cellculture for application to cells.

(II) Imaging of a Cell or Intracellular Molecule Using a GlucoseDerivative

The target cell to be imaged using the glucose derivative of the presentinvention is not particularly limited, but can be mammal-derived cells,cells of a microbe such as E. coli or yeast, plant cells, fertilizedeggs, or the like. These cells may be in any form including cellsisolated from a living body, cells present in a tissue isolated from aliving body, cells present in a tissue of a living body, primarycultured cells after isolation from a living body, and established celllines. The cell to be imaged may be a normal cell or may be an abnormalcell (e.g., a cancer cell).

In the imaging method for a cell according to the present invention, thedetection of the glucose derivative of the present invention taken intothe cell can be performed by a commonly-used method for detectingfluorescence. For example, this method can be performed as follows: thedetection of the glucose derivative present within the cell in themethod of the present invention can be performed by measuring thefluorescence of the target cell in advance, subsequently contacting theglucose derivative with the target cell for a given time, washing outthe resultant after the given time, measuring again the fluorescence ofthe target cell, and evaluating an increase in fluorescence intensityrelative to the fluorescence intensity of the target cell before thecontact. Alternatively, the cell may be imaged during the contact withthe glucose derivative using an appropriate apparatus, such as aconfocal microscope, which is capable of discriminating among theinterior of the cell, the cell membrane, and the exterior of the cell.By recognizing the fluorescence intensity as an image, the cellintracellularly containing the glucose derivative of the presentinvention can be imaged, thereby making it possible to detect the cell.The evaluation may be conducted by means of total fluorescence intensityor fluorescence intensity distribution exhibited by a large number ofcells using a fluorescence plate reader, flow cytometry, or the like.

Use of the glucose derivative of the present invention permits celldetection and/or imaging based on fluorescence (e.g., blue color). Theglucose derivative of the present invention can be used at the same timewith a glucose derivative having a different fluorophore, for example,green fluorescence-emitting2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-D-glucose(2-NBDG) or2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-L-glucose(2-NBDLG), and/or red fluorescence-emitting 2-TexasRed-2-amino-2-deoxy-L-glucose (2-TRLG). 2-NBDG, 2-NBDLG, and 2-TRLG aredescribed in WO2010/16587 (Patent Literature 2) (the disclosure of whichis incorporated herein by reference). As a result, two-color orthree-color evaluation can be achieved.

For example, 2-NBDLG has the property of being specifically taken into acancer and can be used for the detection of the cancer. The D-glucosederivative of the present invention (e.g., CDG or QDG) can be used atthe same time with 2-NBDLG or 2-TRLG. As a result, the status evaluationof cancer cells or whole tumor cell masses containing cancer cells canalso be conducted.

Specifically, for the imaging of cancer cells, etc., the concurrent useof the green fluorescent L-glucose derivative 2-NBDLG, which isspecifically taken into the cancer cells, with the cellmembrane-impermeable red fluorescent 2-TRLG is effective for examiningnonspecific uptake ascribable to membrane injury, inflammation, or thelike (see WO2012/133688 (Patent Literature 4) filed by the presentinventors). In addition, if there is a method capable of visualizingnormal cells present in the neighborhood of a cancer, or non-cancercells that are not yet malignant but in a premalignant state, moreaccurate and highly reliable information on the nature of the cancer isobtained. A candidate compound therefor is a D-glucose derivative thatemits blue fluorescence and is taken into cells via GLUT, to which theD-glucose derivative of the present invention (e.g., CDG or QDG) belong.

(III) Detection or Imaging of a Cancer Cell Using an L-GlucoseDerivative

One embodiment of the L-glucose derivative of the present invention is acompound in which a particular fluorescence-emitting molecule having acoumarin backbone or quinoline backbone is bound to L-glucose having theproperty of being not taken into normal cells.

The uptake of the blue D-glucose derivative CDG or QDG and the blueL-glucose derivative CLG or QLG of the present invention into cancercells showed D form-dominant uptake similar to the relationship betweenthe green D-glucose derivative 2-NBDG and the green L-glucose derivative2-NBDLG. An experiment using the blue L-glucose derivative of thepresent invention, for example, CLG confirmed a specific uptake intocancer cells. Thus, the L-glucose derivative of the present inventioncan also be used for specific detection of cancer cells.

In the method of the present invention, types of cancer cells that canbe detected are not particularly limited, but include, for example,cancer cells of cancer of the ophthalmologic field such as eyelids andlacrimal glands, cancer of ears such as external ears and middle ears,cancers of nose such as nasal cavities and paranasal cavities, lungcancer, oral cancer, larynx cancer, throat cancer, esophageal cancer,stomach cancer, cancer of digestive organs such as small intestine andlarge intestine, cancer of the gynecological field including breastcancer, uterine cancer, ovary cancer, and fallopian tube cancer, cancerof reproductive organs, kidney cancer, bladder cancer, prostate cancer,anus cancer, skin cancer, bone cancer, muscular cancer (sarcoma), bloodcancer such as leukemia, malignant lymphoma, peripheral and centralnervous cancers, and glial cancer.

In the method for detecting a cancer according to the present invention,the L-glucose derivative of the present invention (e.g., CLG or QLG) canbe used at the same time with an additional fluorescently labeledglucose derivative, for example, 2-NBDG, 2-NBDLG, or 2-TRLG, or acombination thereof. As a result, the status evaluation of cancer cellsor whole tumor cell masses containing cancer cells can also beconducted.

The method for detecting a cancer according to the present invention andthe imaging agent therefor can be used for determination of the presenceof tumor cells, the status evaluation thereof, or the discriminationthereof from normal cells with respect to tissues excised at the time ofsurgery, oral tumors, endoscopically obtained digestive organ tumors,gynecologic tumors such as uterine cervix cancer, or biopsy preparationsobtained at the time of biopsy diagnosis, etc., of the lung or variousorgans. As a result, detailed cell evaluation at a cell level can beconducted rapidly with a convenient apparatus equipped withfluorescence. This is effective, for example, for determining aguideline for the selection of a treatment method, determining thetherapeutic effects of drugs or the like, and determining theappropriate extent of surgery after exposure of an affected part.

In the detection method of the present invention, the detection of theL-glucose derivative present within the cancer cell can be performed,for example, by measuring the fluorescence of the target cell inadvance, subsequently contacting the fluorescently labeled L-glucosederivative with the target cell for a given time, washing out theresultant after the given time, measuring again the fluorescence of thetarget cell, and evaluating an increase in fluorescence intensityrelative to the fluorescence intensity of the target cell before thecontact. By recognizing the fluorescence intensity as an image, the cellintracellularly containing the L-glucose derivative can be imaged,thereby making it possible to detect the cancer cell or the cell at arisk thereof. The evaluation may be conducted by means of totalfluorescence intensity or fluorescence intensity distribution exhibitedby a large number of cells using a fluorescence plate reader, flowcytometry, or the like.

The L-glucose derivative of the present invention, when administered toa vascular vessel such as the vein, permits systemic imaging. Inaddition, the L-glucose derivative, when locally administered to atissue to be observed, permits cell imaging.

In addition, in the case of using the radiolabeled L-glucose derivativeof the present invention, a cancer can also be detected by PET.

As is evident from the above description, the glucose derivative of thepresent invention is useful for detecting cancer cells and also usefulas, for example, an active ingredient for an imaging agent forvisualizing cancer cells. The L-glucose derivative may be dissolved in asolvent for dissolution thereof (injectable physiological saline, etc.)and provided in the form of a solution, or may be combined with asolvent for dissolution thereof and provided in the form of a kit forpreparing a solution by dissolution when used. The concentration of thefluorescent L-glucose derivative in the solution may be adjusted in therange of, for example, 1 nM to 100 mM. The method for detecting a cancercell using the L-glucose derivative of the present invention may be usedin combination with a method known per se in the art to further improvedetermination accuracy.

EXAMPLES

Hereinafter, the present invention will be described in detail withreference to Examples. However, the present invention is not construedas being limited by the description below.

Synthesis of Glucose Derivative

Example 1: Synthesis of CDG

CDG was synthesized from D-glucosamine hydrochloride as follows:

(1-1) Synthesis of 2-oxo-2H-chromen-7-yl trifluoromethanesulfonateRepresented by Following Formula

In an argon atmosphere, 4-methylumbelliferone (100 mg, 617 μmol) wasdissolved in pyridine (6 mL), and the solution was cooled in ice.Trifluoromethanesulfonic anhydride (114 μL, 679 μmol) was added dropwisethereto. Then, the mixture was stirred at room temperature for 5 hours.After the completion of the reaction, the reaction mixture was subjectedto extraction with ethyl acetate, and the extract was washed withsaturated saline. The organic layer was dried over anhydrous sodiumsulfate, and the solvent was distilled off. The residue was purifiedusing a silica gel column (SiO₂, 20 g) to obtain the compound (160 mg,88%) as a white solid.

(1-2) Synthesis of2-deoxy-2-(2,2,2-trichloroethoxycarbonylamino)-D-glucose Represented byFollowing Formula

D-Glucosamine hydrochloride (10 g, 46.4 mmol) was dissolved in water(100 mL). To the solution, NaHCO₃ (10 g, 119 mmol) was added at roomtemperature. Then, 2,2,2-trichloroethyl chloroformate (7.7 mL, 55.7mmol) was added dropwise thereto at 0° C. The mixture was stirred at 0°C. for 20 minutes, gradually heated to room temperature, and stirredovernight. The deposited crystals were collected by filtration andwashed with water and ether. After overnight drying, the obtained whitecrystals were recrystallized from warm methanol, and the obtainedcrystals were collected by filtration to obtain the compound (6.0 g,36%).

(1-3) Synthesis of methyl4,6-O-benzylidene-2-deoxy-2-(2,2,2-trichloroethoxycarbonylamino)-α-D-glucopyranosideRepresented by Following Formula

In an argon atmosphere,2-deoxy-2-(2,2,2-trichloroethoxycarbonylamino)-D-glucose (6.0 g, 16.9mmol) was suspended in methanol (200 mL). To the suspension,chlorotrimethylsilane (21 mL) was added dropwise at 0° C. After thedropwise addition, the mixture was heated to reflux and stirred for 2hours. After the completion of the reaction, the reaction mixture wasallowed to cool, and the solvent was distilled off under reducedpressure. Dimethylformamide (200 mL) was added to the residue, thenbenzylidene dimethyl acetal (4.2 mL) and (±)-camphorsulfonic acid (110mg) were added, and the mixture was stirred overnight at roomtemperature. After the completion of the reaction, the reaction mixturewas neutralized by the addition of a saturated aqueous solution ofsodium bicarbonate. Acetonitrile was distilled off under reducedpressure until the solution volume became 0.5 L. Then, the residue wassubjected to extraction with ethyl acetate, and the extract was washedwith a saturated aqueous solution of sodium bicarbonate and then washedwith saturated saline. The organic layer was dried over sodium sulfate,and the solvent was distilled off. The residue was purified using asilica gel column (SiO₂, 75 g) to obtain the compound (1.0 g, 12%) aswhite crystals.

(1-4) Synthesis of methyl4,6-O-benzylidene-2-deoxy-2-amino-α-D-glucopyranoside Represented byFollowing Formula

In an argon atmosphere, methyl4,6-O-benzylidene-2-deoxy-2-(2,2,2-trichloroethoxycarbonylamino)-α-D-glucopyranoside(1.0 g, 2.19 mmol) was dissolved in acetic acid (20 mL). To thesolution, zinc (1.0 g) was added, and the mixture was stirred for 3.5hours. After the completion of the reaction, the reaction mixture wasfiltered, and the solvent was distilled off. The residue wasfreeze-dried from dioxane to obtain the compound (600 mg, 98%) as whitecrystals.

(1-5) Synthesis of methyl4,6-O-benzylidene-2-deoxy-2-(2-oxo-2H-chromen-7-yl)amino-α-D-glucopyranosideRepresented by Following Formula

In an argon atmosphere, toluene (3.4 mL) was added to activatedmolecular sieve 4A (pellet), methyl4,6-O-benzylidene-2-deoxy-2-amino-α-D-glucopyranoside (116.0 mg, 340μmol), 2-oxo-2H-chromen-7-yl trifluoromethanesulfonate (100 mg, 340μmol), tBuXPhos Pd G1(chloro[2-(di-tert-butylphosphino)-2′,4′,6′-triisopropyl-1,1′-biphenyl][2-(2-aminoethyl)phenyl)]palladium(II))(Sigma-Aldrich) (31.1 mg, 34 μmol), tBuXPhos(2-di-tert-butylphosphino-2′,4′,6′-triisopropylbiphenyl) (Sigma-Aldrich)(39.3 mg, 68 μmol), and Cs₂CO₃ (276.9 mg, 850 μmol), and the mixture wasrefluxed for 4 hours. After the completion of the reaction, the reactionmixture was subjected to extraction with ethyl acetate, and the extractwas washed with saturated saline. The organic layer was dried overanhydrous sodium sulfate, and the solvent was distilled off. The residuewas purified using a silica gel column (SiO₂, 5 g) to obtain thecompound (51.2 mg, 35%) as a pale yellow solid.

(1-6) Synthesis of 2-deoxy-2-(2-oxo-2H-chromen-7-yl)amino-D-glucose(CDG) Represented by Following Formula

The compound methyl4,6-O-benzylidene-2-deoxy-2-(2-oxo-2H-chromen-7-yl)amino-α-D-glucopyranoside(40 mg, 94 μmol) was dissolved in an 80% aqueous acetic acid solution (2mL), and the solution was heated to 50° C. and stirred 2 hours. Afterthe completion of the reaction, the solvent was distilled off. 6 Nhydrochloric acid (1.8 mL) was added to the residue, and the mixture washeated to 80° C. and stirred for 4 hours. After the completion of thereaction, the solvent was distilled off. The residue was dissolved indimethylformamide. The solution was purified by reverse-phase HPLC. Afraction containing the compound of interest was freeze-dried to obtainthe compound as a pale yellow solid.

The yield was 9.6 mg, and the percent yield was 47%. Also, results ofanalyzing the obtained compound are as follows.

¹H NMR (D₂O, 400 MHz): δ 7.75 (dd, J=1.37 Hz, 9.61 Hz, 1H, Coumarin-4H),δ 7.30 (d, J=8.69 Hz, 0.7H, Coumarin-5Hα) δ 7.29 (d, J=8.69 Hz, 0.3H,Coumarin-5Hβ), δ 6.69-6.65 (m, 1H, Coumarin-6H), δ 6.59 (s, 1H,Coumarin-8H), δ 6.01 (d, J=9.60 Hz, 0.7H, Coumarin-3Hα), δ 6.01 (d,J=9.61 Hz, 0.3H, Coumarin-3Hβ), δ 5.20 (d, J=3.55 Hz, 0.7H, H-1α), δ3.78-3.21 (m, 6H, H-2, H-3, H-4, H-5, H-6, H-6′); ESI-MS: C₁₅H₁₈NO₇[M+H]⁺ calc.: 324.1, found: 324.0. maximum excitation wavelength (Exmax) 366.5 nm, maximum fluorescence wavelength (Em max) 454.5 nm.

Example 2: Synthesis of CLG

CLG was synthesized in the same way as in CDG using L-glucosaminehydrochloride instead of D-glucosamine hydrochloride. The yield was 13.2mg, and the percent yield was 32%. Also, results of analyzing theobtained compound are as follows.

¹H NMR (D₂O, 400 MHz): δ 7.75 (dd, J=1.83 Hz, 9.15 Hz, 1H, Coumarin-4H),δ 7.31 (d, J=8.69 Hz, 0.6H, Coumarin-5Hα) δ 7.30 (d, J=8.69 Hz, 0.4H,Coumarin-5Hβ), β 6.69-6.65 (m, 1H, Coumarin-6H), δ 6.60 (s, 1H,Coumarin-8H), δ 6.02 (d, J=9.61 Hz, 0.6H, Coumarin-3Hα), δ 6.01 (d,J=9.15 Hz, 0.4H, Coumarin-3Hβ), δ 5.21 (d, J=3.20 Hz, 0.7H, H-1α), δ3.82-3.22 (m, 6H, H-2, H-3, H-4, H-5, H-6, H-6′); ESI-MS: C₁₅H₁₈NO₇[M+H]⁺ calc.: 324.1, found: 324.1.

Example 3: Synthesis of QDG

QDG was synthesized from D-glucosamine hydrochloride as follows:

(3-1) Synthesis of N-(3-iodophenyl)cinnamamide Represented by FollowingFormula

m-Iodoaniline (10 g, 45.7 mmol) and potassium carbonate (9.47 g, 68.6mmol) were suspended in acetone/water (1/2, 94 mL). To the suspension,cinnamic chloride (9.3 g, 56.2 mmol) was added dropwise, and the mixturewas stirred for 30 minutes. The reaction was terminated by pouring toice water. The deposited white solid was collected by filtration andwashed with water and diethyl ether. The solid was dried overnight usinga pump to obtain the compound (17.6 g, quant) as white crystals.

(3-2) Synthesis of 7-iodoquinolin-2(1H)-one Represented by FollowingFormula

In an argon atmosphere, the compound N-(3-iodophenyl)cinnamamide (1.0 g,2.86 mmol) was dissolved in chlorobenzene (28.6 mL), and the solutionwas cooled in ice. Aluminum chloride (1.91 g, 14.3 mmol) was addedthereto, and the mixture was heated to 100° C. and stirred for 2 hours.The reaction was terminated by pouring to ice water. The reactionmixture was separated and washed with ethyl acetate (300 mL) andsaturated saline (200 mL×3). The organic layer was dried over anhydroussodium sulfate, and the solvent was distilled off. The residue waspurified using a silica gel column (SiO₂, 30 g) to obtain the compound(300 mg, 39%) as a pink solid.

(3-3) Synthesis of methyl4,6-O-benzylidene-2-deoxy-2-(2-oxo-1,2-dihydroquinoline-7-yl)amino-α-D-glucopyranosideRepresented by Following Formula

In an argon atmosphere, dioxane (1.05 mL) was added to activatedmolecular sieve 4A (pellet), methyl4,6-O-benzylidene-2-deoxy-2-(2-oxo-2H-chromen-7-yl)amino-α-D-glucopyranoside(30.3 mg, 76.7 μmol), 7-iodoquinolin-2(1H)-one (4.6 mg, 51.2 μmol),tBuXPhos Pd G1(chloro[2-(di-tert-butylphosphino)-2′,4′,6′-triisopropyl-1,1′-biphenyl][2-(2-aminoethyl)phenyl)]palladium(II))(3.6 mg, 5.12 μmol) (Sigma-Aldrich), tBuXPhos(2-di-tert-butylphosphino-2′,4′,6′-triisopropylbiphenyl) (4.2 mg, 10.23μmol) (Sigma-Aldrich), and tBuONa (49.2 mg, 512 μmol), and the mixturewas refluxed for 1 hour. After the completion of the reaction, thereaction mixture was subjected to extraction with ethyl acetate, and theextract was washed with saturated saline. The organic layer was driedover anhydrous sodium sulfate, and the solvent was distilled off. Theresidue was purified using a silica gel column (SiO₂, 5 g) to obtain thecompound (18.3 mg, 84%) as a pale yellow solid.

(3-4) Synthesis of2-deoxy-2-(2-oxo-1,2-dihydroquinolin-7-yl)amino-D-glucose (QDG)Represented by Following Formula

The compound methyl4,6-O-benzylidene-2-deoxy-2-(2-oxo-1,2-dihydroquinolin-7-yl)amino-α-D-glucopyranoside(46 mg, 108 μmol) was dissolved in an 80% aqueous acetic acid solution(2 mL), and the solution was heated to 50° C. and stirred for 2 hours.After the completion of the reaction, the solvent was distilled off. 6 Nhydrochloric acid (1.8 mL) was added to the residue, and the mixture washeated to 80° C. and stirred for 4 hours. After the completion of thereaction, the solvent was distilled off. The residue was dissolved inwater. The solution was purified by reverse-phase HPLC. A fractioncontaining the compound of interest was freeze-dried to obtain thecompound (18.9 mg, 53%) as a pale yellow solid. The yield was 18.9 mg,and the percent yield was 53%. Also, results of analyzing the obtainedcompound are as follows.

¹H NMR (D₂O, 400 MHz): δ 7.78 (d, J=9.15 Hz, 0.6H, Quinolinone-4Hα), δ7.77 (d, J=9.61 Hz, 0.4H, Quinolinone-4Hβ), δ 7.38 (t, J=8.69 Hz, 1H,Quinolinone-5H), δ 6.72-6.67 (td, J=2.29 Hz, J=10.1 Hz, 1H,Quinolinone-6H), δ 6.51 (d, J=2.29 Hz, 0.4H, Quinolinone-8Hβ), δ 6.49(d, J=1.83 Hz, 0.6H, Quinolinone-8Hα), δ 6.24 (d, J=9.15 Hz, 0.6H,Quinolinone-3Hα), δ 6.22 (d, J=9.15 Hz, 0.4H, Quinolinone-3Hβ), δ 5.21(d, J=3.20 Hz, 0.6H, H-1α), δ 3.82-3.29 (m, 6H, H-2, H-3, H-4, H-5, H-6,H-6′); ESI-MS: C₁₅H₁₈N₂O₆ [M+H]⁺ calc.: 323.1, found: 323.1. maximumexcitation wavelength (Ex max) 353.5 nm, maximum fluorescence wavelength(Em max) 423.0 nm.

Example 4: Synthesis of QLG

QLG was synthesized in the same way as in QDG using L-glucosaminehydrochloride instead of D-glucosamine hydrochloride. The yield was 23.2mg, and the percent yield was 33%. Also, results of analyzing theobtained compound are as follows.

¹H NMR (D₂O, 400 MHz): δ 7.77 (d, J=9.15 Hz, 0.5H, Quinolinone-4Hα), δ7.76 (d, J=9.61 Hz, 0.5H, Quinolinone-4Hβ), δ 7.40-7.35 (m, 1H,Quinolinone-5H), δ 6.71-6.67 (td, J=2.29 Hz, J=7.78 Hz, 1H,Quinolinone-6H), δ 6.51-6.48 (m, 1H, Quinolinone-8H), δ 6.25-6.20 (m,1H, Quinolinone-3H), δ 5.22 (d, J=3.66 Hz, 0.5H, H-1α), δ 3.83-3.29 (m,6H, H-2, H-3, H-4, H-5, H-6, H-6′); ESI-MS: C₁₅H₁₈N₂O₆ [M+H]⁺ calc.:323.1, found: 323.1.

Example 5: Synthesis of 4-MCDG

4-MCDG was synthesized as follows:

(5-1) Synthesis of 2-oxo-4-methyl-2H-chromen-7-yltrifluoromethanesulfonate Represented by Following Formula

In an argon atmosphere, 4-methylumbelliferone (1 g, 5.68 mmol) wasdissolved in pyridine (30 mL), and the solution was cooled in ice.Trifluoromethanesulfonic anhydride (1.0 μL, 6.24 mmol) was addeddropwise thereto. Then, the mixture was stirred at room temperature for1 hour. After the completion of the reaction, the reaction mixture wassubjected to extraction with ethyl acetate, and the extract was washedwith saturated saline. The organic layer was dried over anhydrous sodiumsulfate, and the solvent was distilled off. The residue was purifiedusing a silica gel column (SiO₂, 30 g) to obtain the compound (1.7 g,97%) as a white solid.

(5-2) Synthesis of methyl4,6-O-benzylidene-2-deoxy-2-(2-oxo-4-Methyl-2H-chromen-7-yl)amino-α-D-glucopyranosideRepresented by Following Formula

In an argon atmosphere, toluene (6.5 mL) was added to activatedmolecular sieve 4A (pellet), methyl4,6-O-benzylidene-2-deoxy-2-amino-α-D-glucopyranoside (166 mg, 0.487mmol), 2-oxo-4-methyl-2H-chromen-7-yl trifluoromethanesulfonate (100 mg,0.324 mmol), SPhos Pd G1(chloro(2-dicyclohexylphosphino-2′,6′-dimethoxy-1,1′-biphenyl)[2-(2-aminoethylphenyl)]palladium(II))(73.9 mg, 97.2 μmol) (Sigma-Aldrich), SPhos(2-dicyclohexylphosphino-2′,6′-dimethoxybiphenyl) (39.9 mg, 97.2 μmol)(Sigma-Aldrich), and Cs₂CO₃ (264 mg, 810 μmol), and the mixture wasrefluxed for 6 hours. After the completion of the reaction, the reactionmixture was subjected to extraction with ethyl acetate, and the extractwas washed with saturated saline. The organic layer was dried overanhydrous sodium sulfate, and the solvent was distilled off. The residuewas purified using a silica gel column (SiO₂, 40 g) to obtain thecompound (47 mg, 33%) as a pale yellow solid.

(5-3) Synthesis of2-deoxy-2-(2-oxo-4-methyl-2H-chromen-7-yl)amino-D-glucose (MCDG)Represented by Following Formula

The compound methyl4,6-O-benzylidene-2-deoxy-2-(2-oxo-4-methyl-2H-chromen-7-yl)amino-α-D-glucopyranoside(65 mg, 148 μmol) was dissolved in an 80% aqueous acetic acid solution(2 mL), and the solution was heated to 50° C. and stirred for 2 hours.After the completion of the reaction, the solvent was distilled off. 6 Nhydrochloric acid (2 mL) was added to the residue, and the mixture washeated to 80° C. and stirred for 12 hours. After the completion of thereaction, the solvent was distilled off. The residue was dissolved inwater. The solution was purified by reverse-phase HPLC. A fractioncontaining the compound of interest was freeze-dried to obtain thecompound as a pale yellow solid. The yield was 26 mg, and the percentyield was 52%. Also, results of analyzing the obtained compound are asfollows.

¹H NMR (CD₃OD, 400 MHz): δ 7.46 (d, J=8.69 Hz, 0.7H, Coumarin-5Hα), δ7.46 (d, J=9.61 Hz, 0.3H, Coumarin-5Hβ), δ 6.77-6.74 (m, 1H,Coumarin-6H), δ 6.66 (d, J=2.29 Hz, 0.3H, Coumarin-8Hβ), δ 6.61 (d,J=2.29 Hz, 0.7H, Coumarin-8Hα), δ 5.92 (d, J=1.37 Hz, 0.7H,Coumarin-3Hα), δ 5.91 (d, J=0.92 Hz, 0.3H, Coumarin-3Hβ), δ 5.18 (d,J=3.20 Hz, 0.7H, H-1α), δ 4.56 (d, J=7.78 Hz, 0.3H, H-1β), δ 3.90-3.33(m, 6H, H-2, H-3, H-4, H-5, H-6, H-6′) δ 2.37 (s, 3H, Me); ESI-MS:C₁₆H₁₉NO₇ [M+H]⁺ calc.: 338.1, found: 338.1. maximum excitationwavelength (Ex max) 362.0 nm, maximum fluorescence wavelength (Em max)446.0 nm.

4-MCLG can be synthesized in the same way as in 4-MCDG usingL-glucosamine.

Example 6: Synthesis of 3-MCDG

3-MCDG was synthesized as follows:

(6-1) Synthesis of 2-oxo-3-methyl-2H-chromen-7-yltrifluoromethanesulfonate Represented by Following Formula

2,4-Dihydrobenzaldehyde (1 g, 7.24 mmol), propionic anhydride (2.5 mL,19.2 mmol), sodium propionate (1.5 g, 15.6 mmol), and piperidine (0.7mL, 9.54 mmol) were added to a reaction vessel and reacted at 160° C.for 1.5 hours. Purified oil was dissolved in methanol, and water wasadded to the solution. The deposited solid was collected by filtration.The compound of interest was recovered from the filtrate. The compoundwas purified using a silica gel column (SiO₂, 55 g). In an argonatmosphere, the obtained crude product (110 mg, 624 μmol) was dissolvedin pyridine (6.24 mL), and the solution was cooled in ice.Trifluoromethanesulfonic anhydride (115 μL, 687 μmol) was added dropwisethereto. Then, the mixture was stirred at room temperature for 1 hour.After the completion of the reaction, the reaction mixture was subjectedto extraction with ethyl acetate, and the extract was washed withsaturated saline. The organic layer was dried over anhydrous sodiumsulfate, and the solvent was distilled off. The residue was purifiedusing a silica gel column (SiO₂, 20 g) to obtain the compound (120 mg,62%) as a white solid.

(6-1) Synthesis of methyl4,6-O-benzylidene-2-deoxy-2-(2-oxo-3-methyl-2H-chromen-7-yl)amino-α-D-glucopyranosideRepresented by Following Formula

In an argon atmosphere, toluene (0.81 mL) was added to activatedmolecular sieve 4A (pellet), methyl4,6-O-benzylidene-2-deoxy-2-amino-α-D-glucopyranoside (33.2 mg, 97.3μmol), 2-oxo-3-methyl-2H-chromen-7-yl trifluoromethanesulfonate (25 mg,81.1 μmol), Pd₂(dba)₃ (tris(dibenzylideneacetone)dipalladium(0)) (7.4mg, 8.11 μmol), BrettPhos(2-(dicyclohexylphosphino)-3,6-dimethoxy-2′,4′,6′-triisopropyl-1,1′-biphenyl)(Sigma-Aldrich) (8.7 mg, 16.2 μmol), and Cs₂CO₃ (26.4 mg, 81.1 μmol),and the mixture was refluxed for 1 hour. After the completion of thereaction, the reaction mixture was subjected to extraction with ethylacetate, and the extract was washed with saturated saline. The organiclayer was dried over anhydrous sodium sulfate, and the solvent wasdistilled off. The residue was purified using a silica gel column (SiO₂,16 g) to obtain the compound (31.2 mg, 88%) as a pale yellow solid.

(6-3) Synthesis of2-deoxy-2-(2-oxo-3-methyl-2H-chromen-7-yl)amino-D-glucose (3-MCDG)Represented by Following Formula

The compound methyl4,6-O-benzylidene-2-deoxy-2-(2-oxo-3-methyl-2H-chromen-7-yl)amino-α-D-glucopyranoside(139 mg, 31.6 μmol) was dissolved in an 80% aqueous acetic acid solution(3 mL), and the solution was heated to 50° C. and stirred for 4 hours.After the completion of the reaction, the solvent was distilled off. 6 Nhydrochloric acid (3 mL) was added to the residue, and the mixture washeated to 80° C. and stirred for 21 hours. After the completion of thereaction, the solvent was distilled off. The residue was dissolved indimethylformamide. The solution was purified by reverse-phase HPLC. Afraction containing the compound of interest was freeze-dried to obtainthe compound as a pale yellow solid.

The yield was 33.2 mg, and the percent yield was 31%. Also, results ofanalyzing the obtained compound are as follows.

¹H NMR (CD₃OD, 400 MHz): δ 7.55 (s, 1H, Coumarin-4H), δ 7.23 (d, J=8.23Hz, 0.7H, Coumarin-5Hα), δ 7.20 (d, J=8.70 Hz, 0.3H, Coumarin-5Hβ), β6.72-6.69 (m, 1H, Coumarin-6H), δ 6.65 (d, J=1.83 Hz, 0.3H,Coumarin-8Hβ), δ 6.60 (d, J=1.83 Hz, 0.7H, Coumarin-8Hα), δ 5.18 (d,J=3.20 Hz, 0.7H, H-1α), δ 4.55 (d, J=8.24 Hz, 0.3H, H-1β), δ 3.90-3.78(m, 3.7H, H-3α, H-5, H-6, H-6′), δ 3.51-3.33 (m, 2H, H-2α, H-3β, H-4), δ3.25 (dd, J=1.37 Hz, 8.23 Hz, 0.3H, H-2β), δ 2.06 (s, 3H, Me); ESI-MS:C₁₆H₂₀NO₇ [M+H]⁺ calc.: 338.1, found: 338.1. maximum excitationwavelength (Ex max) 361.0 nm, maximum fluorescence wavelength (Em max)460.0 nm.

3-MCLG can be synthesized in the same way as in 3-MCDG usingL-glucosamine.

Example 7: Synthesis of 4-TFMCDG

4-TFMCDG was synthesized as follows:

(7-1) Synthesis of 2-oxo-2H-4-trifluoromethyl-chromen-7-yltrifluoromethanesulfonate Represented by Following Formula

In an argon atmosphere, 4-trifluoromethylumbelliferone (0.2 g, 0.869mmol) was dissolved in pyridine (4.3 mL), and the solution was cooled inice. Trifluoromethanesulfonic anhydride (0.16 mL, 0.956 mmol) was addeddropwise thereto. Then, the mixture was stirred at room temperature for2 hours. After the completion of the reaction, the reaction mixture wassubjected to extraction with ethyl acetate, and the extract was washedwith 1 N hydrochloric acid and saturated saline. The organic layer wasdried over anhydrous sodium sulfate, and the solvent was distilled off.The residue was purified using a silica gel column (SiO₂, 40 g) toobtain the compound (285 mg, 90%) as a white solid.

(7-2) Synthesis of methyl4,6-O-benzylidene-2-deoxy-2-(2-oxo-2H-4-trifluoromethyl-chromen-7-yl)amino-α-D-glucopyranosideRepresented by Following Formula

In an argon atmosphere, toluene (5.5 mL) was added to activatedmolecular sieve 4A (pellet), methyl4,6-O-benzylidene-2-deoxy-2-amino-α-D-glucopyranoside (141.4 mg, 0.414mmol), 2-oxo-2H-4-trifluoromethyl-chromen-7-yl trifluoromethanesulfonate(100 mg, 0.276 mmol), SPhos Pd G1(chloro(2-dicyclohexylphosphino-2′,6′-dimethoxy-1,1′-biphenyl)[2-(2-aminoethylphenyl)]palladium(II))(63 mg, 82.8 μmol) (Sigma-Aldrich), SPhos(2-dicyclohexylphosphino-2′,6′-dimethoxybiphenyl) (34 mg, 82.8 μmol)(Sigma-Aldrich), and Cs₂CO₃ (224.8 mg, 690 μmol), and the mixture wasrefluxed for 2 hours. After the completion of the reaction, the reactionmixture was subjected to extraction with ethyl acetate, and the extractwas washed with 1 N hydrochloric acid and saturated saline. The organiclayer was dried over anhydrous sodium sulfate, and the solvent wasdistilled off. The residue was purified using a silica gel column (SiO₂,40 g) to obtain the compound (42.3 mg, 31%) as a pale yellow solid.

(7-3) Synthesis of2-deoxy-2-(2-oxo-2H-4-trifluoromethyl-chromen-7-yl)amino-D-glucose(4-TFMCDG) Represented by Following Formula

The compound methyl4,6-O-benzylidene-2-deoxy-2-(2-oxo-2H-4-trifluoromethyl-chromen-7-yl)amino-α-D-glucopyranoside(42 mg, 85.1 μmol) was dissolved in an 80% aqueous acetic acid solution(2 mL), and the solution was heated to 50° C. and stirred for 2 hours.After the completion of the reaction, the solvent was distilled off. 6 Nhydrochloric acid (2 mL) was added to the residue, and the mixture washeated to 80° C. and stirred for 3 hours. After the completion of thereaction, the solvent was distilled off. The residue was dissolved inwater. The solution was purified by reverse-phase HPLC. A fractioncontaining the compound of interest was freeze-dried to obtain thecompound as a yellow solid. The yield was 18 mg, and the percent yieldwas 54%. Also, results of analyzing the obtained compound are asfollows.

¹H NMR (CD₃OD, 400 MHz): δ 7.43-7.38 (m, 1H, Coumarin-5H), δ 6.79 (dd,J=2.29 Hz, J=9.15 Hz, 0.7H, Coumarin-5Hα), δ 6.78 (dd, J=2.29 Hz, J=8.69Hz, 0.3H, Coumarin-5Hβ), δ 6.72 (d, J=2.29 Hz, 0.3H, Coumarin-6Hβ), δ6.69 (d, J=2.29 Hz, 0.7H, Coumarin-6Hα), δ 6.37 (s, 0.7H, Coumarin-8Hα),δ 6.35 (s, 0.3H, Coumarin-8Hβ), δ 5.18 (d, J=3.20 Hz, 0.7H, H-1α), δ4.57 (d, J=8.24 Hz, 0.3H, H-1β), δ 3.90-3.33 (m, 6H, H-2, H-3, H-4, H-5,H-6, H-6′); ESI-MS: C₁₆H₁₆F₃NO₇ [M+H]⁺ calc.: 392.1, found: 392.1.maximum excitation wavelength (Ex max) 380.0 nm, maximum fluorescencewavelength (Em max) 500.0 nm.

Example 8: Synthesis of 3-TFMCDG

3-TFMCDG was synthesized as follows:

(8-1) Synthesis of 2-oxo-3-trifluoromethyl-2H-chromen-7-yltrifluoromethanesulfonate Represented by Following Formula

Acetic acid (17.5 mL) was added to 2-oxo-2H-chromen-7-yltrifluoromethanesulfonate (500 mg, 1.71 mmol), sodiumtrifluoromethanesulfinate (400 mg, 2.56 mmol), and manganese(III)acetate dihydrate (914 mg, 3.41 mmol), and the mixture was stirred atroom temperature. After 19 hours, sodium trifluoromethanesulfinate (400mg, 2.56 mmol) and manganese(III) acetate dihydrate (914 mg, 3.41 mmol)were further added thereto. After another 3 hours, sodiumtrifluoromethanesulfinate (400 mg, 2.56 mmol) was further added thereto.26.5 hours after the start of the reaction, the reaction was terminatedby the addition of water. The reaction mixture was subjected toextraction with ethyl acetate, and the extract was washed with saturatedsaline. The organic layer was dried over anhydrous sodium sulfate, andthe solvent was distilled off. The residue was purified using a silicagel column (SiO₂, 40 g) to obtain the compound (258.5 mg, 42%) as awhite solid.

(8-2) Synthesis of methyl4,6-O-benzylidene-2-deoxy-2-(2-oxo-3-trifluoromethyl-2H-chromen-7-yl)amino-α-D-glucopyranosideRepresented by Following Formula

In an argon atmosphere, toluene (0.97 mL) was added to activatedmolecular sieve 4A (pellet), methyl4,6-O-benzylidene-2-deoxy-2-amino-α-D-glucopyranoside (39.6 mg, 116μmol), 2-oxo-3-trifluoromethyl-2H-chromen-7-yl trifluoromethanesulfonate(35 mg, 96.6 μmol), Pd₂(dba)₃ (tris(dibenzylideneacetone)dipalladium(0))(8.8 mg, 9.66 μmol), BrettPhos(2-(dicyclohexylphosphino)-3,6-dimethoxy-2′,4′,6′-triisopropyl-1,1′-biphenyl)(Sigma-Aldrich) (10.4 mg, 19.3 μmol), and Cs₂CO₃ (31.4 mg, 96.6 μmol),and the mixture was refluxed for 1 hour. After the completion of thereaction, the reaction mixture was subjected to extraction with ethylacetate, and the extract was washed with saturated saline. The organiclayer was dried over anhydrous sodium sulfate, and the solvent wasdistilled off. The residue was purified using a silica gel column (SiO₂,16 g) to obtain the compound (13.8 mg, 29%) as a pale yellow solid.

(8-3) Synthesis of2-deoxy-2-(2-oxo-3-trifluoromethyl-2H-chromen-7-yl)amino-D-glucose(3-TFMCDG) Represented by Following Formula

The compound methyl4,6-O-benzylidene-2-deoxy-2-(2-oxo-3-trifluoromethyl-2H-chromen-7-yl)amino-α-D-glucopyranoside(40 mg, 94 μmol) was dissolved in an 80% aqueous acetic acid solution (3mL), and the solution was heated to 50° C. and stirred for 6 hours.After the completion of the reaction, the solvent was distilled off. 6 Nhydrochloric acid (3 mL) was added to the residue, and the mixture washeated to 80° C. and stirred for 5 hours. After the completion of thereaction, the solvent was distilled off. The residue was dissolved indimethylformamide. The solution was purified by reverse-phase HPLC. Afraction containing the compound of interest was freeze-dried to obtainthe compound as a pale yellow solid.

The yield was 23.2 mg, and the percent yield was 27%. Also, results ofanalyzing the obtained compound are as follows.

¹H NMR (CD₃OD, 400 MHz): δ 8.17 (s, 1H, Coumarin-4H), δ 7.41 (d, J=8.69Hz, 0.7H, Coumarin-5Hα) δ 7.40 (d, J=8.69 Hz, 0.3H, Coumarin-5Hβ), β6.80-6.76 (m, 1H, Coumarin-6H), δ 6.66 (d, J=1.83 Hz, 0.3H,Coumarin-8Hβ), δ 6.64 (d, J=2.29 Hz, 0.7H, Coumarin-8Hα), δ 5.18 (d,J=3.20 Hz, 0.7H, H-1α), δ 4.58 (d, J=7.78 Hz, 0.3H, H-1β), δ 3.91-3.68(m, H-3, H-5α, H-6, H-6′), δ 3.58 (dd, J=10.1 Hz, 3.20 Hz, 0.7H, H-2α),δ 3.49-3.34 (m, 2.6H, H-2β, H-4, H-5β); ESI-MS: C₁₆H₁₇F₃NO₇ [M+H]⁺calc.: 392.1, found: 392.1. maximum excitation wavelength (Ex max) 381.0nm, maximum fluorescence wavelength (Em max) 455.0 nm.

Example 9: Synthesis of 6-F-CDG

6-F-CDG was synthesized as follows:

(9-1) Synthesis of methyl2-deoxy-2-(2-oxo-2H-chromen-7-yl)amino-α-D-glucopyranoside Representedby Following Formula

The compound methyl4,6-O-benzylidene-2-deoxy-2-(2-oxo-2H-chromen-7-yl)amino-α-D-glucopyranoside(200 mg, 470.1 μmol) was dissolved in an 80% aqueous acetic acidsolution (5 mL), and the solution was heated to 50° C. and stirred for 2hours. After the completion of the reaction, the solvent was distilledoff. The residue was freeze-dried from 1,4-dioxane to obtain thecompound (159 mg, quant.) as a pale yellow solid.

(9-2) Synthesis of methyl3,4-di-O-benzoyl-2-deoxy-2-(2-oxo-2H-chromen-7-yl)amino-α-D-glucopyranosideRepresented by Following Formula

In an argon atmosphere, methyl2-deoxy-2-(2-oxo-2H-chromen-7-yl)amino-α-D-glucopyranoside (50 mg, 148μmol) was dissolved by the addition of pyridine (1.5 mL). To thesolution, triphenylmethyl chloride (206 mg, 740 μmol) was added, and themixture was heated to 60° C. After 2 hours, the reaction mixture wasallowed to cool, neutralized with a saturated aqueous solution of sodiumbicarbonate, and subjected to extraction with ethyl acetate, and theextract was washed with a saturated aqueous solution of sodiumbicarbonate and saturated saline. The organic layer was dried overanhydrous sodium sulfate, and the solvent was distilled off. In an argonatmosphere, the obtained residue was dissolved by the addition ofpyridine (1.5 mL). To the solution, benzoyl chloride (140 μL, 1.205mmol) was added, and the mixture was heated to 40° C. After 2 hours, thereaction mixture was subjected to extraction with ethyl acetate, and theextract was washed with a saturated aqueous solution of sodiumbicarbonate and saturated saline. The organic layer was dried overanhydrous sodium sulfate, and the solvent was distilled off. An ice-cold90% aqueous trifluoroacetic acid solution (1.5 mL) was added to theobtained residue. After 30 minutes, the mixture was diluted withchloroform and washed with a saturated aqueous solution of sodiumbicarbonate and saturated saline. The organic layer was dried overanhydrous sodium sulfate, and the solvent was distilled off. The residuewas purified using a silica gel column (SiO₂, 40 g) to obtain thecompound (47.7 mg, 59%) as a pale yellow solid.

(9-3) Synthesis of methyl3,4-di-O-benzoyl-2,6-dideoxy-6-fluoro-2-(2-oxo-2H-chromen-7-yl)amino-α-D-glucopyranosideRepresented by Following Formula

In an argon atmosphere, methyl2-deoxy-2-(2-oxo-2H-chromen-7-yl)amino-α-D-glucopyranoside (220 mg, 403μmol) was dissolved in dichloromethane (10 mL), and the solution wascooled to −17° C. N,N-Diethylaminosulfur trifluoride (320 μL, 2.42 mmol)was added dropwise thereto, and the mixture was heated to roomtemperature. After 2 hours, the mixture was cooled to −17° C.N,N-Diethylaminosulfur trifluoride (320 μL, 2.42 mmol) was further addeddropwise thereto, and the mixture was heated to room temperature. 4hours after the start of the reaction, the reaction mixture was cooledin ice, and the reaction was terminated by the addition of methanol. Thereaction mixture was subjected to extraction with ethyl acetate, and theextract was washed with saturated saline. The organic layer was driedover anhydrous sodium sulfate, and the solvent was distilled off. Theresidue was purified using a silica gel column (SiO₂, 40 g) to obtainthe compound (104 mg, 47%) as a pale yellow solid.

(9-4) Synthesis of2,6-dideoxy-6-fluoro-2-(2-oxo-2H-chromen-7-yl)amino-D-glucose (6-F-CDG)Represented by Following Formula

Acetic acid (1 mL) and 6 N hydrochloric acid (20 mL) were added to thecompound methyl3,4-di-O-benzoyl-2,6-dideoxy-6-fluoro-2-(2-oxo-2H-chromen-7-yl)amino-α-D-glucopyranoside(104 mg, 190 μmol), and the mixture was heated to 80° C. and stirred for4 days. Then, the solvent was distilled off. Trifluoroacetic acid (5 mL)and 6 N hydrochloric acid (10 mL) were added to the residue, and themixture was heated to 80° C. and stirred for 3 days. After thecompletion of the reaction, the solvent was distilled off. The residuewas dissolved in dimethylformamide. The solution was purified byreverse-phase HPLC. A fraction containing the compound of interest wasfreeze-dried to obtain the compound as a pale yellow solid.

The yield was 10.2 mg, and the percent yield was 17%. Also, results ofanalyzing the obtained compound are as follows.

¹H NMR (CD₃OD, 400 MHz): δ 7.74 (d, J=9.15 Hz, 0.7H, Coumarin-4Hα), δ7.74 (d, J=9.15 Hz, 0.3H, Coumarin-4Hβ), δ 7.30 (d, J=8.69 Hz, 0.7H,Coumarin-5Hα), δ 7.27 (d, J=8.24 Hz, 0.3H, Coumarin-5Hβ), δ 6.75-6.72(m, 1H, Coumarin-6H), δ 6.66 (d, J=1.83 Hz, 0.3H, Coumarin-8Hβ), δ 6.62(d, J=2.29 Hz, 0.7H, Coumarin-8Hα), δ 6.00 (d, J=9.15 Hz, 0.7H,Coumarin-3Hα), δ 5.98 (d, J=9.15 Hz, 0.3H, Coumarin-3Hβ), δ 5.19 (d,J=3.20 Hz, 0.7H, H-1α), δ 4.75-4.51 (m, 2.2H, H-1β, H-3β, H-5β, H-6,H-6′β), δ 3.97 (dddd, J=27.5 Hz, 10.1 Hz, 4.1 Hz, 1.3 Hz, 0.7H, H-6′α),δ 3.78 (t, J=9.61 Hz, 0.7H, H-3α), δ 3.55-3.42 (m, 2.4H, H-2α, H-4,H-5α); ESI-MS: C₁₅H₁₇FNO₆ [M+H]⁺ calc.: 326.1, found: 326.1.

Example 10: Synthesis of 4-F-CDG

4-F-CDG was synthesized as follows:

(10-1) Synthesis of methyl2-deoxy-2-(2-oxo-2H-chromen-7-yl)amino-3,6-di-O-pivaloyl-α-D-glucopyranosideRepresented by Following Formula

In an argon atmosphere, methyl2-deoxy-2-(2-oxo-2H-chromen-7-yl)amino-α-D-glucopyranoside (435 mg, 1.29mmol) was dissolved by the addition of pyridine (6.5 mL) anddichloromethane (6.5 mL), and the solution was cooled in ice. Pivaloylchloride (565 μL, 4.59 mmol) was added dropwise thereto, and the mixturewas heated to room temperature. After 1 hour, the reaction wasterminated by the dropwise addition of a saturated aqueous solution ofsodium bicarbonate. The reaction mixture was subjected to extractionwith ethyl acetate, and the extract was washed with a saturated aqueoussolution of sodium bicarbonate and saturated saline. The organic layerwas dried over anhydrous sodium sulfate, and the solvent was distilledoff. The residue was purified using a silica gel column (SiO₂, 40 g) toobtain the compound (431.8 mg, 66%) as a pale yellow solid.

(10-2) Synthesis of methyl2-deoxy-2-(2-oxo-2H-chromen-7-yl)amino-3,6-di-O-pivaloyl-α-D-galactopyranosideRepresented by Following Formula

In an argon atmosphere, methyl2-deoxy-2-(2-oxo-2H-chromen-7-yl)amino-3,6-di-O-pivaloyl-α-D-glucopyranoside(150 mg, 297 μmol) was dissolved in dichloromethane (10 mL) and pyridine(1 mL), and the solution was cooled in ice. Trifluoromethanesulfonicanhydride (74 μL, 445 μmol) was added dropwise thereto, and the mixturewas heated to room temperature. After 1.5 hours, the reaction mixturewas subjected to extraction with ethyl acetate, and the extract waswashed with a saturated aqueous solution of sodium bicarbonate andsaturated saline. The organic layer was dried over anhydrous sodiumsulfate, and the solvent was distilled off. In an argon atmosphere, theresidue was dissolved by the addition of dimethylformamide (3 mL), andthe solution was cooled in ice. Sodium nitrite (71.7 mg, 69 μmol) wasadded thereto. After 1.5 hours, the reaction solution was purified witha silica gel column (SiO₂, 16 g). The compound (79.5 mg, 53%) wasobtained as a pale yellow solid.

(10-3) Synthesis of methyl2,4-dideoxy-4-fluoro-2-(2-oxo-2H-chromen-7-yl)amino-3,6-di-O-pivaloyl-α-D-glucopyranosideRepresented by Following Formula

In an argon atmosphere, methyl2-deoxy-2-(2-oxo-2H-chromen-7-yl)amino-3,6-di-O-pivaloyl-α-D-galactopyranoside(225 mg, 445 μmol) was dissolved in dichloromethane (8.9 mL), and thesolution was cooled to −40° C. N,N-Diethylaminosulfur trifluoride (352.8μL, 2.67 mmol) was added dropwise thereto, and the mixture was heated toroom temperature. 4 hours after the start of the reaction, the reactionmixture was cooled to −20° C., and the reaction was terminated by theaddition of methanol. After extraction with chloroform, the extract waswashed with saturated saline. The organic layer was dried over anhydroussodium sulfate, and the solvent was distilled off. The residue waspurified using a silica gel column (SiO₂, 40 g) to obtain the compound(174.7 mg, 77%) as a pale yellow solid.

(10-4) Synthesis of2,4-dideoxy-4-fluoro-2-(2-oxo-2H-chromen-7-yl)amino-D-glucose (4-F-CDG)Represented by Following Formula

The compound methyl2,4-dideoxy-4-fluoro-2-(2-oxo-2H-chromen-7-yl)amino-3,6-di-O-pivaloyl-α-D-glucopyranoside(170 mg, 335 μmol) was dissolved in a solution of 4.5 N hydrogenchloride in dioxane (6 mL) and 6 N hydrochloric acid (3 mL), and thesolution was heated to 80° C. and stirred for 6 hours. After thecompletion of the reaction, the solvent was distilled off. The residuewas dissolved in dimethylformamide. The solution was purified byreverse-phase HPLC. A fraction containing the compound of interest wasfreeze-dried to obtain the compound as a pale yellow solid.

The yield was 8.4 mg, and the percent yield was 8%. Also, results ofanalyzing the obtained compound are as follows. ¹H NMR (CD₃OD, 400 MHz):δ 7.75 (d, J=9.61 Hz, 1H, Coumarin-4H), δ 7.30 (d, J=8.69 Hz, 1H,Coumarin-5H), δ 6.75 (dd, J=8.24 Hz, 1.83 Hz, 1H, Coumarin-6H), δ 6.67(d, J=1.83 Hz, 0.3H, Coumarin-8Hβ), δ 6.63 (d, J=2.29 Hz, 0.7H,Coumarin-8Hα), δ 6.01 (d, J=9.15 Hz, 0.7H, Coumarin-3Hα), δ 5.99 (d,J=9.15 Hz, 0.3H, Coumarin-3Hβ), δ 5.18 (t, J=3.20 Hz, 0.7H, H-1α), δ4.61 (d, J=7.78 Hz, 0.3H, H-1β), δ 4.40 (ddd, J=51.2 Hz, 8.69 Hz, 1.37Hz, 0.7H, H-4α), δ 4.35 (ddd, J=50.8 Hz, 8.69 Hz, 0.92 Hz, 0.3H, H-4β),δ 4.06-3.98 (m, 1.7H, H-3α, H-6), δ 3.88-3.48 (m, 2.6H, H-2β, H-3β, H-5,H-6′), δ 3.36 (d, J=10.1 Hz, 0.7H, H-2α); ESI-MS: C₁₅H₁₇FNO₆ [M+H]⁺calc.: 326.1, found: 326.1.

(7) Fluorescence Spectrum

The fluorescence spectra of CDG synthesized in (1) and QDG synthesizedin (2) were compared with the fluorescence spectrum of 2-NBDLG(manufactured by Peptide Institute, Inc.). The results are shown in FIG.1.

The numeric values described above the graph denote the fluorescencemaximum of CDG and QDG, respectively. It is evident that thefluorescence maximum was largely shifted to shorter wavelength ascompared with the fluorescence maximum of 2-NBDLG present around 550 nm.Thus, even if CDG (or CLG that exhibits the same fluorescence spectrumas CDG) or QDG (or QLG that exhibits the same fluorescence spectrum asQDG) can be used at the same time with 2-NBDLG (or 2-NBDG that exhibitsthe same fluorescence spectrum as 2-NBDLG), these derivatives can beeasily distinguished from each other based on the difference influorescence wavelength (the principal spectrum of 2-NBDLG was presentat 500 nm or greater, and a small peak and valley on the spectrum seenat 500 nm or smaller were influenced by excitation light).

Example 11: Influence of Cytochalasin B (CB) on Increase in FluorescenceIntensity Caused by Administration of CDG to Mouse Insulinoma Cell(MIN6)

Increase in fluorescence intensity caused by the administration of CDG,and the influence of a glucose transporter (GLUT) inhibitor CB thereonwere confirmed by targeting MIN6 cells.

(Experimental Method)

(1) Preparation of Mouse Insulinoma Cell (MIN6)

A 96-well clear-bottom plate having wells of 8 rows (A to H) and 12columns (1 to 12) (μClear-PLATE, Greiner Bio-One, BLACK) was used inmeasurement. A culture solution containing MIN6 cells suspended at adensity of 60×10⁴ cells/mL was added dropwise at 10 μL/well to thecentral portion of the well, and then, the plate was left standing for40 minutes in an incubator for fixation. The cells were cultured by theaddition of a culture solution at 200 μL/well (6000 cells/well). Forlayout, the cells were seeded to wells B to H on the third column andwells A to G on the fifth column. Medium replacement was performed byreplacing half the amount with a fresh one once every two days at 0 to 4DIV (days in vitro) and every day at 5 DIV or later. The cells weresubjected to the experiment at culture days 10 to 15 (10 to 15 DIV).Well A on the third column and well H on the fifth column were used ascontrols for checking whether or not washout was accurately performedwithout seeding the cells. The wells on the fourth column were used ascell-free blanks using only a Krebs-Ringer buffer solution (KRB,containing 0.1 mM gap junction inhibitor carbenoxolone and 5.6 mMglucose).

(1-1) Culture of MIN6 Cell

The MIN6 cells used were cells kindly provided by professor JunichiMiyazaki (Osaka University) and subcultured 6 to 10 times. Half theamount of the culture solution was replaced with a fresh one once everytwo days.

(1-2) Composition of Culture Solution Used in Culture of MIN6 Cell

13.4 g of high glucose-containing Dulbecco's modified Eagle's Medium(DMEM-HG) (Sigma-Aldrich, #D5648), 3.4 g of NaHCO₃, and 5 μL of2-mercaptoethanol were dissolved in 1 L of ultrapure water, and the pHof the solution was adjusted to 7.30 to 7.35 in a CO₂ incubator of 37°C. The culture solution was supplemented with fetal bovine serum(Hyclone, Cat# SH30070.03) at a final concentration of 10% andpenicillin-streptomycin at a final concentration of 0.5%.

(2) Preparation of CDG Solution and Additional Fluorescent SugarDerivative

Preparation of CDG Solution

The whole amount of the 0.5 mg CDG vial was recovered using 0.73 mL intotal of 10% dimethyl sulfoxide (DMSO) and dissolved by addition to 7.0ml of a KRB solution for image acquisition according to the methoddescribed in Non Patent Literature 2. The solution was applied at afinal concentration of 100 μM to the cells.

Preparation of 2-NBDG Solution

The whole amount of one 0.5 mg 2-NBDG vial was dissolved in 1.83 mL of aKRB solution for data acquisition. The solution was applied at a finalconcentration of 200 μM to the cells.

(2-1) KRB Solution for Data Acquisition

Composition of Volume Used in Data Acquisition with FluorescenceMicroplate Reader

129.0 mM NaCl, 4.75 mM KCl, 1.19 mM KH₂PO₄, 1.19 mM MgSO₄.7H₂O, 1.0 mMCaCl₂.2H₂O, 5.02 mM NaHCO₃, 5.6 mM D-glucose, and 10 mM HEPES (the pHwas adjusted to 7.35 with 1 M NaOH). 0.1 mM carbenoxolone(Sigma-Aldrich, #C4790) was added thereto for the purpose of inhibitingthe entrance and exit of fluorescently labeled glucose by way of gapjunction/hemichannel. This KRB solution for data acquisition was used asa solution for preparing the CDG solution and the additional fluorescentsugar derivative solution.

(3) Fluorescence Measurement

CDG and 2-NBDG were each independently administered to the wells on thethird and fifth columns using an 8-channel pipette such that thesederivatives were alternately placed on the same plate. Before theadministration, the autofluorescence of each well was measured inadvance using a fluorescence microplate reader (Flex Station, MolecularDevices, LLC). The measurement conditions involved Ex of 367 nm, Em of455 nm, and cut off of 420 nm on the CDG detector side (blue channel)and Ex of 470 nm, Em of 540 nm, and cut off of 495 nm on the 2-NBDGdetector side (green channel). The measurement was conducted at BottomRead, Averaging 3, and Photomultiplier sensitivity high. Well Scan Modewas used as a measurement method. The Well Scan Mode each independentlymeasures 9 observation regions (diameter: 1.5 mm) divided from one well.

Further, CB (final concentration: 10 μM) was preliminarily administeredto wells for the measurement of the effect of the glucose transportinhibitor cytochalasin B (CB) from 2 minutes before the administrationof CDG, and KRB was added to the other wells. CDG and 2-NBDG wereadministered at 37° C. for 5 minutes.

After the completion of the administration, the operation of dilutingthe fluorescent solution in each well using 300 of a KRB solution for 30seconds was repeated at the predetermined number of times. The number ofrepetitions was determined such that fluorescence intensity exhibited bywell A on the third column and well H on the fifth column selected as acontrol group was at the same level as the fluorescence intensity of thecell-free blank wells. Complete washout was confirmed in each run of theexperiment. In the case of CDG and 2-NBDG, this washout process required8 minutes. Therefore, fluorescence measurement was carried out 9 minutesafter the administration.

According to this method, even if cells having disrupted membraneintegrity have temporarily taken CDG and 2-NBDG therein after contactwith these compounds, the compounds are already drained to the outsideof the cells and washed out at the time of measurement. Therefore, theircontribution to increase in fluorescence intensity in the wholeobservation area was judged as being ignorable.

The effect of the glucose transport inhibitor on change in fluorescenceintensity caused by the application of the D-glucose derivative (CDG) tothe MIN6 cells at culture day 13 was confirmed by use of the methoddescribed above. Increase in the fluorescence intensity of the cells wasmeasured in the presence and absence of CB (10 μM). The results areshown in FIG. 2A.

In the presence of CB, the fluorescence intensity was largely attenuatedas compared with the absence of the inhibitor, suggesting thatGLUT-mediated uptake contributes not a little to the cellular uptake ofCDG. The numeral within the parentheses denotes the number of effectiveobservation regions. Two similar experiments were independentlyconducted and both produced similar results. The fluorescence intensitywas decreased to 47.3% and 42.9%, respectively, on average in thepresence of CB relative to the absence of CB.

The unpaired t-test or the ANOVA and Bonferroni-Dunn test was used inthe statistics (the same holds true for the description below).

Example 12: Influence of Phloretin (PHT) on Increase in FluorescenceIntensity Caused by Administration of CDG to MIN6 Cell

Increase in fluorescence intensity caused by the administration of CDGto the MIN6 cells at culture day 13, and the influence of PHTfunctioning as a GLUT inhibitor and a water channel inhibitor thereonwere confirmed in the same way as in Example 11. The results are shownin FIG. 2B.

PHT (final concentration: 150 μM) was preliminarily administered towells for the measurement of the effect of PHT from 1 minute before theadministration of CDG, and KRB was added to the other wells. CDG and2-NBDG were administered at 37° C. for 5 minutes.

In the presence of PHT, the fluorescence intensity was remarkablyattenuated as compared with the absence of the inhibitor. Two similarexperiments were independently conducted and both produced similarresults. The fluorescence intensity was decreased to 28.5% and 28.8%,respectively, on average in the presence of PHT relative to the absenceof PHT. The degree of this inhibitory effect of PHT was stronger thanthe inhibitory effect of CB shown in FIG. 2A, suggesting the possibilitythat a pathway other than GLUT is also involved, together with GLUT, inthe cellular uptake of CDG.

Example 13: Influence of Phloretin (PHT) on Increase in FluorescenceIntensity Caused by Administration of CDG or CLG to MIN6 Cell

Increase in fluorescence intensity caused by the administration of CDGor CLG to the MIN6 cells at culture day 15, and the inhibitory effect ofPHT thereon were compared on the same dish.

MIN6 cells on one 96-well dish were targeted where there werePHT-containing wells and PHT-free wells to which CDG and CLG werealternately administered. The administration solutions were concurrentlyadministered using an 8-channel pipette. The dish was left standing for5 minutes, and then, washout was also performed concurrently for thewells. Change in fluorescence intensity was measured on each observationregion. The results are shown in FIG. 2C.

As a result of the experiment, increase in fluorescence intensity causedby the administration of the D-glucose derivative CDG was significantlyinhibited by PHT serving as a GLUT inhibitor and a water channelinhibitor, as in the results of Example 12. Also, PHT significantlyinhibited the uptake of the L-glucose derivative CLG.

It is to be noted that the fluorescence intensity was still increasednot a little by the administration of CDG even in the presence of PHT,and the degree of this increase was almost the same as the degree of theincrease in fluorescence intensity by CLG.

Furthermore, the increase in fluorescence intensity caused by theadministration of the L-glucose derivative CLG was significantly smallerthan the increase in fluorescence intensity caused by the administrationof the D-glucose derivative CDG. This quantitatively demonstrated thatthe uptake of the blue fluorescent glucose derivatives CDG and CLG intomouse insulinoma MIN6 cells exhibits D/L stereoselectivity.

A previous case of quantitatively comparing fluorescent glucosederivatives taken into cells while exhibiting D/L stereoselectivity isthe report of the present inventors as to the administration of thegreen glucose derivatives 2-NBDG and 2-NBDLG to MIN6 cells. In thiscase, the cellular uptake of the L-glucose derivative 2-NBDLG is alwayssmaller than the cellular uptake of the D-glucose derivative 2-NBDG (seeWO2012/133688 (Patent Literature 4)). D form-dominant uptake was alsofound in the combination of the blue D-glucose derivative CDG and theblue L-glucose derivative CLG of the present invention, suggesting thepossibility of reflecting difference in the three-dimensional structureof glucose.

Comparative Example: Influence of Cytochalasin B (CB) or Phloretin (PHT)on Increase in Fluorescence Intensity Caused by Administration of GreenFluorescent D-Glucose Derivative 2-NBDG to MIN6 Cell

The inhibitory effect of CB (10 μM) or PHT (150 μM) on increase influorescence intensity caused by the administration of the greenfluorescent D-glucose derivative 2-NBDG was confirmed. On the same dayas the experiment of Example 11, the same MIN6 cell series at 13 daysafter the start of culture was used, and the green fluorescent D-glucosederivative 2-NBDG was administered thereto to examine the inhibitoryeffect of CB (10 μM) or PHT (150 μM) on increase in fluorescenceintensity.

Specifically, a KRB solution containing 200 μM 2-NBDG was administeredto the cells, which were then left standing for 5 minute, followed bywashout. Increase in the fluorescence intensity of the cells betweenbefore and after the administration was measured. Each observationregion was measured three times at an excitation light wavelength of 470nm, a fluorescence acquisition wavelength of 540 nm, and a cutoff filterof 495 nm, and a mean thereof was calculated. The results are shown inFIG. 3.

The uptake of 2-NBDG into the MIN6 cells was significantly inhibited byCB and more strongly inhibited by PHT, like the previous report of theinventors (WO2012/133688 (Patent Literature 4)). Two experimentsindependently carried out both produced similar results. Thefluorescence intensity was decreased to 30.8% and 36.4%, respectively,on average in the presence of CB relative to the absence of CB. Thefluorescence intensity was decreased to 18.6% and 15.7%, respectively,on average in the presence of PHT relative to the absence of PHT. Theseresults are similar to the inhibitory effects of CB and PHT on increasein fluorescence intensity caused by the administration of CDG as seen inFIGS. 2A and 2B. However, the uptake of 2-NBDG was remarkably attenuatedin the presence of PHT, whereas the fluorescence intensity remained nota little even in the presence of PHT by the administration of CDG.

In this way, the comparison between 2-NBDG and CDG can offer informationon the influence of difference in fluorescent group bound to D-glucoseon cellular uptake, and important hints about transport pathways.

Example 14: Influence of Excessive D-Glucose or Excessive L-Glucose onIncrease in Fluorescence Intensity Caused by Administration of CDG toMIN6 Cell

Whether or not increase in fluorescence intensity caused by theadministration of the blue fluorescent D-glucose derivative CDG (100 μM)to the mouse insulinoma cells MIN6 at culture day 13 was inhibited by alarge excess of D-glucose or L-glucose was examined. The bluefluorescent L-glucose derivative CLG was similarly analyzed as acontrol. The results are shown in FIG. 4. However, the followingconditions were changed.

(Experimental method)

(1) Preparation of KRB Solution Dedicated to Excessive GlucoseAdministration Group

Although the usual KRB solution contains 129 mM NaCl and 5.6 mMD-glucose, the osmotic pressure of a control solution (non-excessiveglucose administration group solution) for this experiment was adjustedto 270 mOsm by substituting a portion of NaCl with choline-Cl, in orderto equalize charge and osmotic pressure between the control solution anda KRB solution for excessive glucose administration. Also, the KRBsolution for excessive glucose administration was allowed to containNaCl and glucose according to the composition given below. In thisrespect, choline-Cl was added thereto in order to equalize osmoticpressure between this solution and the control solution (non-excessiveglucose administration group solution).

100 mM NaCl and 5.6 mM D-glucose (non-excessive glucose administrationgroup solution)

100 mM NaCl and 50 mM D-glucose (50 mM excessive D-glucoseadministration group solution)

100 mM NaCl, 50 mM L-glucose, and 5.6 mM D-glucose (50 mM excessiveL-glucose administration group solution)

These dedicated KRB solutions were also used as a solution for preparingCDG and CLG solutions in this Example.

(2) Fluorescence Measurement

CDG and CLG were placed on different plates for the experiment. A CDGsolution (or a CLG solution) prepared using each dedicated KRB solutionwas administered thereto using an 8-channel pipette (37° C., 5 min).Before the administration, replacement with each dedicated KRB solutionwas carried out in advance, and the autofluorescence of each well wasmeasured with a fluorescence microplate reader. The time from the startof the replacement to immediately before the administration wasapproximately 28 minutes.

After the completion of the administration, first, the fluorescentsolution in each well was temporarily diluted using 200 μL of thededicated KRB solution and then diluted using 300 μL of the usual KRBsolution.

The cellular uptake of D-glucose via GLUT present in the cell membranerequires that D-glucose should bind to a D-glucose-binding site in theGLUT protein. Specifically, the cellular uptake is considered to occurunder the mechanism where: D-glucose binds to a D-glucose-binding sitein the GLUT protein initially in an outward-open state in the cellmembrane, and this binding alters the three-dimensional structure ofGLUT to an inward-open state; subsequently, D-glucose is released fromthe binding site in GLUT and eventually transported into the cell fromthe exterior because the D-glucose faces the interior of the cell. Inthis respect, if a large excess of D-glucose is present in the solution,D-glucose occupies D-glucose-binding sites in GLUT. Thus, the transportrate into the cell does not exceed the fixed level and reaches aplateau.

Here, provided that the increase in fluorescence intensity caused by theadministration of the blue fluorescent D-glucose derivative CDG to theMING cells is attributed to the GLUT-mediated cellular uptake of CDG aspredicted from the results of FIG. 2A, it is considered that CDG bindsto a D-glucose-binding site in GLUT prior to passage through GLUT. Thus,in this respect, if a large excess of D-glucose is present in thesolution, D-glucose occupies D-glucose-binding sites in GLUT. Thus, itis predicted that CDG cannot bind to the binding site and the transportof CDG into the cell is also inhibited (competitive inhibition).

The influence of the presence of 50 mM L-glucose was also studied as acontrol in evaluating the influence of the presence of a large excess(50 mM) of D-glucose on increase in fluorescence intensity.

As a result of the experiment, the increase in fluorescence intensitycaused by the administration of CDG was significantly suppressed in thepresence of 50 mM D-glucose as compared with the control in the absenceof D-glucose, but was not suppressed by the addition of 50 mM L-glucose(FIG. 4A). On the other hand, the increase in fluorescence intensitycaused by the administration of CLG was also slightly inhibited by 50 mMD-glucose (FIG. 4B). However, the statistical difference therebetweenwas very slight. In fact, although the significant difference in theANOVA and Bonferroni-Dunn test used in this experiment is regarded asbeing significant at a P value of less than 0.0083, the actual numericvalue was 0.0060, which merely fell below the significance level by anarrow margin. The increase in fluorescence intensity caused by theadministration of CLG was not inhibited by 50 mM L-glucose.

Example 15: Influence of Cytochalasin B (CB) or Phloretin (PHT) onIncrease in Fluorescence Intensity Caused by Administration of QDG toMIN6 Cell

Increase in fluorescence intensity caused by the administration of QDGand the influence of CB or PHT thereon were confirmed in the same way asin Examples 11 and 12 using QDG instead of CDG. The results are shown inFIG. 5.

(1) Preparation of QDG Solution

The whole amount of the 0.5 mg QDG vial was recovered using 0.71 mL intotal of 10% dimethyl sulfoxide (DMSO) and dissolved by addition to 7.1ml of KRB solution for image acquisition according to the methoddescribed in Non Patent Literature 2. The solution was applied at afinal concentration of 100 μM to the cells.

As a result of the experiment, the increase in fluorescence intensitycaused by the administration of QDG was significantly inhibited by CB(FIG. 5A) and more strongly inhibited in the presence of PHT (FIG. 5B).Each observation region was measured three times at an excitation lightwavelength of 354 nm, a fluorescence acquisition wavelength of 435 nm,and a cutoff filter of 420 nm, and a mean of the results was calculated.

Example 16: Imaging of Tumor Cell Mass Consisting of Mouse InsulinomaCells (MIN6) Using CDG/2-NBDLG/2-TRLG

A fluorescent mixed solution containing 100 μM CDG, 100 μM 2-NBDLG, and20 μM 2-TRLG (CDG/2-NBDLG/2-TRLG) was administered at 37° C. for 3minutes to MIN6 cell masses (spheroids) at culture day 17, andfluorescent imaging was conducted.

(Experimental Method)

(1) Preparation of Mouse Insulinoma Cell (MIN6) Spheroid

10 μL of a culture solution containing MIN6 cells suspended at a densityof 6×10⁴ cells/mL was added dropwise onto each glass cover slip, andthen, the glass cover slip was left standing for 40 minutes in anincubator for fixation to the glass surface. The cells were cultured bythe addition of 3 mL of a culture solution (600 cells/slip). Spheroidswere formed by continuing the culture for 15 to 17 days. Half the amountof the culture solution was replaced with a fresh one once every threedays.

(1-1) Culture of MIN6 Cell

The MIN6 cells used were cells kindly provided by professor JunichiMiyazaki (Osaka University) and subcultured 6 to 10 times. Half theamount of the culture solution was replaced with a fresh one once everytwo days.

(1-2) Composition of Culture Solution Used in Culture of MIN6 Cell

13.4 g of high glucose-containing Dulbecco's modified Eagle's Medium(DMEM-HG) (Sigma-Aldrich, #D5648), 3.4 g of NaHCO₃, and 5 μL of2-mercaptoethanol were dissolved in 1 L of ultrapure water, and the pHof the solution was adjusted to 7.30 to 7.35 in a CO₂ incubator of 37°C. The culture solution was supplemented with fetal bovine serum(Hyclone, Cat# SH30070.03) at a final concentration of 10% andpenicillin-streptomycin at a final concentration of 0.5%.

(2) Preparation of CDG Solution and Mixed Solution with AdditionalFluorescent Sugar Derivatives

Preparation of CDG Solution

The whole amount of the 0.5 mg CDG vial was recovered using 0.73 mL intotal of 10% DMSO and dissolved by addition to 7.0 ml of a KRB solutionfor image acquisition according to the method described in Non PatentLiterature 2.

Preparation of 2-NBDLG Solution

The whole amount of one 0.5 mg 2-NBDLG vial was dissolved in 7.3 mL of aKRB solution for image acquisition.

Preparation of 100 μM CDG+100 μM 2-NBDLG+20 μM 2-TRLG Mixed Solution

The CDG solution and the 2-NBDLG solution were mixed at a ratio of 1:1.The whole amount of the 0.2 mg 2-TRLG vial was recovered using 130 μL intotal of DMSO and mixed with 13 ml of the CDG+2-NBDLG mixed solutionaccording to the method described in Non Patent Literature 2 (finalconcentration: 100 μM CDG+100 μM 2-NBDLG+20 μM 2-TRLG mixed solution).

(2-1) KRB Solution for Image Acquisition

The KRB solution for image acquisition used was a solution having thefollowing composition.

129.0 mM NaCl, 4.75 mM KCl, 1.19 mM KH₂PO₄, 1.19 mM MgSO₄.7H₂O, 1.0 mMCaCl₂.2H₂O, 5.02 mM NaHCO₃, 5.6 mM D-glucose, and 10 mM HEPES (the pHwas adjusted to 7.35 with 1 M NaOH). 0.1 mM carbenoxolone(Sigma-Aldrich, #C4790) was added thereto for the purpose of inhibitingthe entrance and exit of fluorescently labeled glucose by way of gapjunction/hemichannel. This KRB solution for data acquisition was usedfor preparing the CDG solution and the mixed solution with theadditional fluorescent sugar derivatives.

(3) Method for Observing MIN6 Cell Using Perfusion Chamber

The glass cover slip with the cultured MIN6 cells was transferred intothe KRB solution for image acquisition in a perfusion chamber loaded ona stage of a fluorescence microscope, followed by measurement.

(3-1) Perfusion Chamber

A silicon plate of 1 mm thickness bored to have a streamline-shaped hole(10 mm wide×35 mm long) was placed via cover glass onto an aluminumheating control platform (Warner Instruments) having a round hole(diameter: 18 mm) for an objective lens at the bottom, and allowed to bein close contact with the cover glass.

The perfusion of the solution was carried out according to the methoddescribed in Non Patent Literature 2.

(4) Perfusate Feeding System to Perfusion Chamber

The KRB solution for image acquisition was warmed in advance in analuminum syringe heater and supplied to the perfusion chamber throughhydrostatic pressure. The perfusion rate was adjusted to 1.3±0.2 mL/minusing a flow rate adjuster. Immediately before being introduced to theperfusion chamber, the solution was rewarmed via an inline heater sothat the actually measured temperature of the perfusate in anobservation region in the chamber was adjusted to 36±1° C. TheCDG/2-NBDLG/2-TRLG mixed solution was able to be supplied in the sameway as above and was switched with the KRB solution for imageacquisition using an electromagnetic valve capable of controlling theopening and closing of the channel. The solution was removed using avacuum pump capable of controlling suction pressure.

(5) Image Acquisition Condition

The fluorescence microscope used was NIKON ECLIPS-Ti, and images wereacquired with a CCD camera Retiga 2000R.

CDG, 2-NBDLG, and 2-TRLG were each detected using the following filtercassette.

CDG (Blue Channel):

Excitation 360/40 nm, Dichroic mirror 400 nm, Emission 460/50 nm.

2-NBDLG (Green Channel):

Excitation 470/40 nm, Dichroic mirror 500 nm, Emission 545/55 nm.

2-TRLG (Red Channel):

Excitation 567/15 nm, Dichroic mirror 593 nm, Emission 593 nm Long pass.

The objective lens used in this method was ×60 oil lens (Plan Fluor60×/0.50-1.25 Oil). The images were acquired at 1600×1200 pixels and adepth of 12 bit.

The results of administering the 100 μM CDG+100 μM 2-NBDLG+20 μM 2-TRLGmixed solution for 3 minutes to the MIN6 cell masses that formedspheroids are shown in FIGS. 6 and 7. FIG. 6 shows fluorescencemicroscopic images taken 6 minutes after the start of washout of themixed solution. FIGS. 6A, 6B, and 6C show blue fluorescence-emittingCDG, green fluorescence-emitting 2-NBDLG, and red fluorescence-emitting2-TRLG, respectively, taken into cells. FIG. 6D is a differentialinterference contrast (DIC) image. The central portion of the spheroidshowed the strong uptake of 2-TRLG, and the cell membrane permeabilitywas enhanced as also described in the previous report of the inventors(WO2012/133688 (Patent Literature 4)). Thus, the uptake of CDG or2-NBDLG exhibited by the cells present in this region seems to be mainlybased on nonspecific uptake ascribable to the enhanced membranepermeability. By contrast, in a doughnut-like region surrounding thecentral portion of the spheroid, only a portion of the cells exhibitedsuch uptake of 2-TRLG, and the uptake of CDG and 2-NBDLG in this regionwas not uniform.

FIG. 7 is a superimposed image of FIGS. 6A and 6B. The uptake of CDG isexpressed in red color instead of blue color in order to facilitatevisualizing the localization of CDG and 2-NBDLG. There existed nearlyred cells that exhibited the relatively strong cellular uptake of CDG,nearly green cells that exhibited the relatively strong cellular uptakeof 2-NBDLG, cells expressed in yellow color as a result of the uptake ofboth CDG and 2-NBDLG, and the like. The results described above indicatethe possibility that the presence of cells that take less 2-NBDLGtherein, but take CDG therein can be detected by the concurrentadministration of CDG and 2-NBDLG to cell masses.

In addition, 2-NBDLG was strongly taken into the cytoplasm to therebyyield an image as if denucleated, whereas CDG was not only taken intothe cytoplasm but unexpectedly taken into the nucleus. This was alsoverified by the presence of cells whose nucleus was stained red byreflecting the uptake of CDG and whose cytoplasm was stained yellow byreflecting the uptake of CDG and 2-NBDLG in FIG. 7.

Example 17: Imaging of Tumor Cell Mass Consisting of Mouse InsulinomaCells (MIN6) Using CLG/2-NBDLG/2-TRLG

Fluorescent imaging was conducted in the same way as in Example 16except that a CLG/2-NBDLG/2-TRLG mixed solution was used instead of theCDG/2-NBDLG/2-TRLG mixed solution for MIN6 cell masses at culture day15.

The results are shown in FIG. 8. FIG. 8 shows fluorescence microscopicimages taken 6 minutes after the start of washout of the 100 μM CLG+100μM 2-NBDLG+20 μM 2-TRLG mixed solution after 3-minute administration ofthe mixed solution to MIN6 cell masses that formed spheroids. FIGS. 8A,8B, and 8C show blue fluorescence-emitting CLG, greenfluorescence-emitting 2-NBDLG, and red fluorescence-emitting 2-TRLG,respectively, taken into cancer cells. FIG. 8D is a differentialinterference contrast (DIC) image. Unlike the case of CDG, all the cellsthat strongly took CLG therein also exhibited the uptake of 2-NBDLG.Like CDG, CLG was also strongly taken not only into the cytoplasm butinto the nucleus.

Example 18: Application of CLG to Acutely Isolated Normal Neuronal Cell

(Experimental Method)

(1) Preparation of CLG Solution and Mixed Solution with AdditionalFluorescent Sugar Derivative

Preparation of CLG Solution

The whole amount of the 0.5 mg CLG vial was recovered using 0.73 mL intotal of 10% DMSO and dissolved (100 μM) by addition to 14.79 ml of aHEPES solution for image acquisition according to the method describedin Non Patent Literature 2.

Preparation of 100 μM CLG+20 μM 2-TRLG Mixed Solution

The whole amount of the 0.2 mg 2-TRLG vial was recovered using 130 μL intotal of DMSO and dissolved in 13 mL of the CLG solution according tothe method described in Non Patent Literature 2 (final concentration:100 μM CLG+20 μM 2-TRLG mixed solution).

(1-1) HEPES Solution for Image Acquisition

A solution having the following composition was used for fluorescenceimage acquisition.

150 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 2 mM CaCl₂, and 10 mM HEPES (the pHof the solution was adjusted to 7.4 with a 1 Mtris(2-amino-2-hydroxymethyl-1,3-propanediol) solution). Theconcentration of glucose was set to 10 mM. 0.1 mM carbenoxolone(Sigma-Aldrich, #C4790) was added thereto for the purpose of inhibitingthe entrance and exit of fluorescently labeled glucose by way of gapjunction/hemichannel. This HEPES solution for image acquisition was usedfor preparing the CLG solution and the mixed solution with theadditional fluorescent sugar derivative.

(2) Observation of Neuronal Cell Using Perfusion Chamber

Neuronal cells of the substantia nigra pars reticulate of the midbrainof a 21-day-old mouse were acutely isolated according to the methoddescribed in the previous reports of the inventors (WO2010/16587 (PatentLiterature 2)) and (WO2012/133688 (Patent Literature 4)) and attached toeach glass cover slip coated with poly-L-lysine. Then, the glass coverslip was transferred into the HEPES solution for image acquisition in aperfusion chamber loaded on a stage of a fluorescence microscope,followed by observation.

(2-1) Perfusion Chamber

A silicon plate of 1 mm thickness bored to have a streamline-shaped hole(10 mm wide×35 mm long) was placed via cover glass onto an aluminumheating control platform (Warner Instruments) having a round hole(diameter: 18 mm) for an objective lens at the bottom, and allowed to bein close contact with the cover glass.

The perfusion of the solution was carried out according to the methoddescribed in Non Patent Literature 2.

(3) Perfusate Feeding System to Perfusion Chamber

The HEPES solution for image acquisition was warmed in advance in analuminum syringe heater and supplied to the perfusion chamber throughhydrostatic pressure. The perfusion rate was adjusted to 1.3±0.2 mL/minusing a flow rate adjuster. Immediately before being introduced to theperfusion chamber, the solution was rewarmed via an inline heater sothat the actually measured temperature of the perfusate in anobservation region in the chamber was adjusted to 32.5±1° C. TheCLG/2-TRLG mixed solution was able to be supplied in the same way asabove and was switched with the HEPES solution for image acquisitionusing an electromagnetic valve capable of controlling the opening andclosing of the channel. The solution was removed using a vacuum pumpcapable of controlling suction pressure.

(4) Image Acquisition Condition

The fluorescence microscope used was NIKON ECLIPS-Ti, and images wereacquired with a CCD camera Retiga 2000R.

CLG and 2-TRLG were each detected using the following filter cassette.

CLG (Blue Channel):

Excitation 360/40 nm, Dichroic mirror 400 nm, Emission 460/50 nm.

2-TRLG (Red Channel):

Excitation 567/15 nm, Dichroic mirror 593 nm, Emission 593 nm Long pass.

The objective lens used in this method was ×20 dry lens (Nikon Plan SFluor). The images were acquired at 1600×1200 pixels.

The results are shown in FIGS. 9 and 10. FIG. 9 shows the case wherechange in fluorescence intensity exhibited by cells between before andafter 3-minute administration of the CLG+2-TRLG mixed solution toacutely isolated normal neuronal cells (arrow) was observed in thewavelength region of blue fluorescence (blue channel) emitted by CLG.The process of background subtraction was conducted both before andafter the administration. FIG. 9A shows the observed autofluorescenceexhibited by the normal neuronal cells (arrow) before the administrationof the mixed solution. FIG. 9B shows a differential interferencecontrast (DIC) image superimposed on the image of FIG. 9A for furtherunderstanding. As is evident, the normal neuronal cells slightly emittedautofluorescence before the administration of the mixed solution. *depicts the debris of killed cells. The debris on the left side of theimage exhibited relatively strong autofluorescence. FIG. 9C is afluorescence microscopic image taken 8 minutes after the start ofwashout of the mixed solution. FIG. 9D is a DIC image superimposed onthe image of FIG. 9C. As seen from the figure, bluefluorescence-emitting CLG was strongly taken into the cell debris (*) ina nonspecific manner, whereas such uptake was not observed in the normalneuronal cells (arrow).

FIG. 10 shows that change in fluorescence between before and after theadministration of the mixed solution in FIG. 9, as observed at the sametime as in the wavelength region of red fluorescence (red channel)emitted by 2-TRLG. FIG. 10A shows an autofluorescence image taken beforethe administration of the mixed solution. FIG. 10B shows a differentialinterference contrast (DIC) image superimposed on the image of FIG. 10Ain order to facilitate visualizing the positions of cells. Theautofluorescence of the cells was very small in the red fluorescencewavelength region. Intense red color at the central portion of the imageresulted from the leakage of a portion of irradiation light to thedetector side through a fluorescence filter because the red fluorescencewas photographed with sensitivity enhanced for the purpose of detectingweak fluorescence (see Dichroic mirror and Emission wavelengths of thered channel). Here, the image is shown as it is without shadingcorrection.

As is evident from FIG. 10C and FIG. 10D which is a DIC imagesuperimposed on the image of FIG. 10C, red fluorescence-emitting 2-TRLGwas taken into the cell debris (*) in a nonspecific manner, but was nottaken into the normal neuronal cells (arrow). In short, nonspecificenhancement in cell membrane permeability that brings about the entry of2-TRLG into the normal neuronal cells indicated by arrows in thisexperiment was not detected, suggesting that the membrane integrity ofthese cells was maintained.

On the other hand, as shown in Example 17, the L-glucose derivative CLGwas strongly taken into the spheroid of the mouse insulinoma cells(MING), which are cancer cells.

Example 19: Application of CDG to Acutely Isolated Normal Neuronal Cell

This experiment was conducted in the same way as the experimental methoddescribed in Example 18 except that CDG was used instead of CLG toprepare a 100 μM CDG+20 μM 2-TRLG mixed solution (concentrations werefinal concentrations) and the neuronal cells of the substantia nigrapars reticulate of the midbrain were prepared from a 14-day-old mouse.

The results are shown in FIGS. 11 and 12. FIG. 11 shows the case wherechange in fluorescence intensity exhibited by cells between before andafter 3-minute administration of the CDG+2-TRLG mixed solution toacutely isolated normal neuronal cells (arrow) was observed in thewavelength region of blue fluorescence (blue channel) emitted by CDG.The process of background subtraction was conducted both before andafter the administration. FIG. 11A shows the observed autofluorescenceexhibited by the normal neuronal cells (arrow) before the administrationof the mixed solution. FIG. 11B shows a differential interferencecontrast (DIC) image superimposed on the image of FIG. 11A for furtherunderstanding. * depicts the debris of killed cells. FIG. 11C is afluorescence microscopic image taken 8 minutes after the start ofwashout of the mixed solution. FIG. 11D is a DIC image superimposed onthe image of FIG. 11C. As seen from the figure, bluefluorescence-emitting CDG was taken into the normal neuronal cells(arrow). On the other hand, such uptake was not observed in the celldebris (*).

FIG. 12 shows that change in fluorescence between before and after theadministration of the mixed solution in FIG. 11, as observed at the sametime in the wavelength region of red fluorescence (red channel) emittedby 2-TRLG. FIG. 12A shows an autofluorescence image taken before theadministration of the mixed solution. FIG. 12B shows a differentialinterference contrast (DIC) image superimposed on the image of FIG. 12Ain order to facilitate visualizing the positions of cells. Theautofluorescence of the cells was very small in the red fluorescencewavelength region.

As is evident from FIG. 12C and FIG. 12D which is a DIC imagesuperimposed on the image of FIG. 12C, red fluorescence-emitting 2-TRLGwas taken into the cell debris (*) in a nonspecific manner, but was nottaken into the normal neuronal cells (arrow). In short, nonspecificenhancement in cell membrane permeability that brings about the entry of2-TRLG into the normal neuronal cells indicated by arrows in thisexperiment was not detected, suggesting that the membrane integrity wasmaintained.

The detailed description above is given merely for illustrating theobjects and subjects of the present invention and is not intended tolimit the scope of the attached claims. It is obvious to those skilledin the art from the instruction described herein that various changesand modification can be made in the described embodiments withoutdeparting from the scope of the attached claims.

INDUSTRIAL APPLICABILITY

The present invention provides a novel glucose derivative. The presentinvention also provides an imaging agent and an imaging method for acell using the glucose derivative.

1. A glucose derivative which is a compound represented by the followingformula (1):

wherein X—Y—Z represents O—C═O, NH—C═O, NR₃—C═O, or N═C—OR₄, wherein R₃represents C₁-C₅ alkyl, and R₄ represents C₁-C₅ alkyl; R₁ and R₂ eachindependently represent a group selected from the group consisting ofhydrogen, halogen, C₁-C₅ alkyl, C₂-C₅ alkenyl, C₂-C₅ alkynyl, C₁-C₅haloalkyl, C₁-C₅ alkylamino, cycloalkyl, phenyl, pyridyl, thiophenyl,pyrrolyl, and furanyl; and G represents a group selected from thefollowing formulas (G1) to (G4):

or a salt thereof.
 2. The glucose derivative according to claim 1, whichis a compound represented by the following formula (2):

wherein X represents O, NH, or NR₃, wherein R₃ represents C₁-C₅ alkyl;and R₁ and R₂ each independently represent a group selected from thegroup consisting of hydrogen, halogen, C₁-C₅ alkyl, C₂-C₅ alkenyl, C₂-C₅alkynyl, C₁-C₅ haloalkyl, C₁-C₅ alkylamino, cycloalkyl, phenyl, pyridyl,thiophenyl, pyrrolyl, and furanyl, or a salt thereof.
 3. (canceled) 4.The glucose derivative according to claim 1, which is a compoundrepresented by the following formula (3):

wherein R₁ and R₂ each independently represent a group selected from thegroup consisting of hydrogen, halogen, C₁-C₅ alkyl, C₂-C₅ alkenyl, C₂-C₅alkynyl, C₁-C₅ haloalkyl, C₁-C₅ alkylamino, cycloalkyl, phenyl, pyridyl,thiophenyl, pyrrolyl, and furanyl; and R₄ represents C₁-C₅ alkyl, or asalt thereof.
 5. The glucose derivative according to claim 1, which is acompound represented by the following formula (4):

wherein X represents O, NH, or NR₃, wherein R₃ represents C₁-C₅ alkyl;and R₁ and R₂ each independently represent a group selected from thegroup consisting of hydrogen, halogen, C₁-C₅ alkyl, C₂-C₅ alkenyl, C₂-C₅alkynyl, C₁-C₅ haloalkyl, C₁-C₅ alkylamino, cycloalkyl, phenyl, pyridyl,thiophenyl, pyrrolyl, and furanyl, or a salt thereof.
 6. (canceled) 7.The glucose derivative according to claim 1, which is a compoundrepresented by the following formula (5):

wherein R₁ and R₂ each independently represent a group selected from thegroup consisting of hydrogen, halogen, C₁-C₅ alkyl, C₂-C₅ alkenyl, C₂-C₅alkynyl, C₁-C₅ haloalkyl, C₁-C₅ alkylamino, cycloalkyl, phenyl, pyridyl,thiophenyl, pyrrolyl, and furanyl; and R₄ represents C₁-C₅ alkyl, or asalt thereof.
 8. The glucose derivative according to claim 1, which isselected from the group consisting of the following compounds:2-deoxy-2-(2-oxo-2H-chromen-7-yl)amino-D-glucose (CDG),2,4-dideoxy-2-(2-oxo-2H-chromen-7-yl)amino-4-fluoro-D-glucose (4-F-CDG),2,6-dideoxy-2-(2-oxo-2H-chromen-7-yl)amino-6-fluoro-D-glucose (6-F-CDG),2-deoxy-2-(2-oxo-1,2-dihydroquinolin-7-yl)amino-D-glucose (QDG),2-deoxy-2-(2-oxo-2H-3-methyl-chromen-7-yl)amino-D-glucose (3-MCDG),2-deoxy-2-(2-oxo-2H-4-methyl-chromen-7-yl)amino-D-glucose (4-MCDG),2-deoxy-2-(2-oxo-2H-3-trifluoromethyl-chromen-7-yl)amino-D-glucose(3-TFMCDG),2-deoxy-2-(2-oxo-2H-4-trifluoromethyl-chromen-7-yl)amino-D-glucose(4-TFMCDG),2-deoxy-2-(2-oxo-1,2-dihydro-3-methyl-quinolin-7-yl)amino-D-glucose(3-MQDG),2-deoxy-2-(2-oxo-1,2-dihydro-4-methyl-quinolin-7-yl)amino-D-glucose(4-MQDG),2-deoxy-2-(2-oxo-1,2-dihydro-3-trifluoromethyl-quinolin-7-yl)amino-D-glucose(3-TFMQDG),2-deoxy-2-(2-oxo-1,2-dihydro-4-trifluoromethyl-quinolin-7-yl)amino-D-glucose(4-TFMQDG), 2-deoxy-2-(2-oxo-2H-chromen-7-yl)amino-L-glucose (CLG),2,4-dideoxy-2-(2-oxo-2H-chromen-7-yl)amino-4-fluoro-L-glucose (4-F-CLG),2,6-dideoxy-2-(2-oxo-2H-chromen-7-yl)amino-6-fluoro-L-glucose (6-F-CLG),2-deoxy-2-(2-oxo-1,2-dihydroquinolin-7-yl)amino-L-glucose (QLG),2-deoxy-2-(2-oxo-2H-3-methyl-chromen-7-yl)amino-L-glucose (3-MCLG),2-deoxy-2-(2-oxo-2H-4-methyl-chromen-7-yl)amino-L-glucose (4-MCLG),2-deoxy-2-(2-oxo-2H-3-trifluoromethyl-chromen-7-yl)amino-L-glucose(3-TFMCLG),2-deoxy-2-(2-oxo-2H-4-trifluoromethyl-chromen-7-yl)amino-L-glucose(4-TFMCLG),2-deoxy-2-(2-oxo-1,2-dihydro-3-methyl-quinolin-7-yl)amino-L-glucose(3-MQLG),2-deoxy-2-(2-oxo-1,2-dihydro-4-methyl-quinolin-7-yl)amino-L-glucose(4-MQLG),2-deoxy-2-(2-oxo-1,2-dihydro-3-trifluoromethyl-quinolin-7-yl)amino-L-glucose(3-TFMQLG), and2-deoxy-2-(2-oxo-1,2-dihydro-4-trifluoromethyl-quinolin-7-yl)amino-L-glucose(4-TFMQLG). 9.-10. (canceled)
 11. A radiolabeled glucose derivativecomprising a glucose derivative according to claim 1, wherein a hydroxygroup at any one of the 2-, 4-, and 6-positions of the glucose issubstituted by ¹⁸F.
 12. The glucose derivative according to claim 1which is in a radiolabeled form selected from2-deoxy-2-(2-oxo-2H-3-[¹¹C]methyl-chromen-7-yl)amino-D-glucose(3-[¹¹C]MCDG),2-deoxy-2-(2-oxo-2H-4-[¹¹C]methyl-chromen-7-yl)amino-D-glucose(4-[¹¹C]MCDG),2-deoxy-2-(2-oxo-2H-3-[¹⁸F]fluorodifluoromethyl-chromen-7-yl)amino-D-glucose(3-[¹⁸F]TFMCDG),2-deoxy-2-(2-oxo-2H-4-[¹⁸F]fluorodifluoromethyl-chromen-7-yl)amino-D-glucose(4-[¹⁸F]TFMCDG),2-deoxy-2-(2-oxo-1,2-dihydro-3-[¹¹C]methyl-quinolin-7-yl)amino-D-glucose(3-[¹¹C]MQDG),2-deoxy-2-(2-oxo-1,2-dihydro-4-[¹¹C]methyl-quinolin-7-yl)amino-D-glucose(4-[¹¹C]MQDG),2-deoxy-2-(2-oxo-1,2-dihydro-3-[¹⁸F]fluorodifluoromethyl-quinolin-7-yl)amino-D-glucose(3-[¹⁸F]TFMQDG),2-deoxy-2-(2-oxo-1,2-dihydro-4-[¹⁸F]fluorodifluoromethyl-quinolin-7-yl)amino-D-glucose(4-[¹⁸F]TFMQDG),2-deoxy-2-(2-oxo-2H-3-[¹¹C]methyl-chromen-7-yl)amino-L-glucose(3-[¹¹C]MCLG),2-deoxy-2-(2-oxo-2H-4-[¹¹C]methyl-chromen-7-yl)amino-L-glucose(4-[¹¹C]MCLG),2-deoxy-2-(2-oxo-2H-3-[¹⁸F]fluorodifluoromethyl-chromen-7-yl)amino-L-glucose(3-[¹⁸F]TFMCLG),2-deoxy-2-(2-oxo-2H-4-[¹⁸F]fluorodifluoromethyl-chromen-7-yl)amino-L-glucose(4-[¹⁸F]TFMCLG),2-deoxy-2-(2-oxo-1,2-dihydro-3-[¹¹C]methyl-quinolin-7-yl)amino-L-glucose(3-[¹¹C]MQLG),2-deoxy-2-(2-oxo-1,2-dihydro-4-[¹¹C]methyl-quinolin-7-yl)amino-L-glucose(4-[¹¹C]MQLG),2-deoxy-2-(2-oxo-1,2-dihydro-3-[¹⁸F]fluorodifluoromethyl-quinolin-7-yl)amino-L-glucose(3-[¹⁸F]TFMQLG),2-deoxy-2-(2-oxo-1,2-dihydro-4-[¹⁸F]fluorodifluoromethyl-quinolin-7-yl)amino-L-glucose(4-[¹⁸F]TFMQLG).
 13. A composition for imaging a target cell, comprisinga glucose derivative according to claim
 1. 14.-20. (canceled)
 21. Amethod for imaging a target cell, comprising the following steps: a.contacting a composition containing a glucose derivative according toclaim 1 with the target cell; and b. detecting fluorescence emitted bythe glucose derivative present within the target cell. 22.-23.(canceled)
 24. The imaging method according to claim 21, wherein thecomposition in the step a further comprises an additional fluorescentlylabeled glucose derivative, and the step b is the step of detecting atleast one of the glucose derivatives present within the target cell.25.-27. (canceled)
 28. A method for detecting a cancer or a cancer cell,comprising the following steps: a. contacting a composition containing aglucose derivative according to claim 1 with a target cell; and b.detecting the glucose derivative present within the target cell, whereinthe glucose derivative is a compound represented by the followingformula (2), (3), (4) or (5) or a salt thereof:

wherein X represents O wherein R₃ represents C₁-C₅ alkyl; and R₁ and R₂independently represent a group selected from the group consisting ofhydrogen, halogen, C₁-C₅ alkyl, C₂-C₅ alkenyl, C₂-C₅ alkynyl, C₁-C₅haloalkyl, C₁-C₅ alkylamino, cycloalkyl, phenyl, pyridyl, thiophenyl,pyrrolyl, and furanyl;

wherein R₁ and R₂ each independently represent a group selected from thegroup consisting of hydrogen, halogen, C₁-C₅ alkyl, C₂-C₅ alkenyl, C₂-C₅alkynyl, C₁-C₅ haloalkyl, C₁-C₅ alkylamino, cycloalkyl, phenyl, pyridyl,thiophenyl, pyrrolyl, and furanyl; and R₄ represents C₁-C₅ alkyl;

wherein X represents O, wherein R₃ represents C₁-C₅ alkyl; and R₁ and R₂each independently represent a group selected from the group consistingof hydrogen, halogen, C₁-C₅ alkyl, C₂-C₅ alkenyl, C₂-C₅ alkynyl, C₁-C₅haloalkyl, C₁-C₅ alkylamino, cycloalkyl, phenyl, pyridyl, thiophenyl,pyrrolyl, and furanyl; or

wherein R₁ and R₂ each independently represents a group selected fromthe group consisting of hydrogen, halogen, C₁-C₅ alkyl, C₂-C₅ alkenyl,C₂-C₅ alkynyl, C₁-C₅ haloalkyl, C₁-C₅ alkylamino, cycloalkyl, phenyl,pyridyl, thiophenyl, pyrrolyl, and furanyl; and R₄ represents C₁-C₅alkyl. 29.-38. (canceled)
 39. An imaging agent for imaging a targetcancer cell, comprising a glucose derivative according to claim 1,wherein the glucose derivative is a compound represented by thefollowing formula (2), (3), (4) or (5) or a salt thereof:

wherein X represents O, wherein R₃ represents C₁-C₅ alkyl; and R₁ and R₂independently represent a group selected from the group consisting ofhydrogen, halogen, C₁-C₅ alkyl, C₂-C₅ alkenyl, C₂-C₅ alkynyl, C₁-C₅haloalkyl, C₁-C₅ alkylamino, cycloalkyl, phenyl, pyridyl, thiophenyl,pyrrolyl, and furanyl;

wherein R₁ and R₂ each independently represent a group selected from thegroup consisting of hydrogen, halogen, C₁-C₅ alkyl, C₂-C₅ alkenyl, C₂-C₅alkynyl, C₁-C₅ haloalkyl, C₁-C₅ alkylamino, cycloalkyl, phenyl, pyridyl,thiophenyl, pyrrolyl, and furanyl; and R₄ represents C₁-C₅ alkyl;

wherein X represents O, wherein R₃ represents C₁-C₅ alkyl; and R₁ and R₂each independently represent a group selected from the group consistingof hydrogen, halogen, C₁-C₅ alkyl, C₂-C₅ alkenyl, C₂-C₅ alkynyl, C₁-C₅haloalkyl, C₁-C₅ alkylamino, cycloalkyl, phenyl, pyridyl, thiophenyl,pyrrolyl, and furanyl; or

wherein R₁ and R₂ each independently represents a group selected fromthe group consisting of hydrogen, halogen, C₁-C₅ alkyl, C₂-C₅ alkenyl,C₂-C₅ alkynyl, C₁-C₅ haloalkyl, C₁-C₅ alkylamino, cycloalkyl, phenyl,pyridyl, thiophenyl, pyrrolyl, and furanyl; and R₄ represents C₁-C₅alkyl. 40.-44. (canceled)